Difference between revisions of "Team:Pasteur Paris/Binding cellulose"

 
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   <p>
 
   <p>
 
  <FONT size="3pt">
 
  <FONT size="3pt">
−
  <a href="#J1">May 25th, 2016</a></br>
+
  <center><a href="#J1">August 24th, 2016</a></center></br>
−
  <a href="#J2">May 26th, 2016</a></br>
+
  <center><a href="#J2">August 25th, 2016</a></center></br>
−
  <a href="#J3">May 27th, 2016</a></br>
+
  <center><a href="#J3">August 29th, 2016</a></center></br>
−
  <a href="#J4">May 28th, 2016</a></br>
+
  <center><a href="#J4">September 12th, 2016</a></center></br>
−
  <a href="#J5">June 3th, 2016</a></br>
+
  <center><a href="#J5">September 14th, 2016</a></center></br>
−
  <a href="#J6">July 7th, 2016</a></br>
+
  <center><a href="#J6">September 29th, 2016</a></center></br>
−
  <a href="#J7">July 22th, 2016</a></br>
+
−
  <a href="#J8">Octo 7th, 2016</a></br>
+
 
</FONT>
 
</FONT>
 
</p>
 
</p>
 
       </center>
 
       </center>
−
     </div>  
+
     </div>
  
−
<h1><B>Chemistry notebook</B></h1>
+
<h1><B>Cellulose binding notebook</B></h1>
−
 
+
<div id="J1"><h2><B>August 24th, 2016 </B></h2></div></br></br>
−
 
+
<h3> <strong>Cellulose binding test </strong></h3>
−
<h2><B>May 25th, 2016 : </B></h2>
+
        </div>
−
<h3> Preparation of cellulose solution: </h3>
+
<div class="text1">
−
<div id="J1">
+
 
<p>
 
<p>
 +
                <U> Aim:</U> This step checks the ability of our protein to bind cellulose.
 +
Moreover, this step will confirm the good design of the protein.</br></br>
  
−
<U> Aim:</U></br> Preparing cellulose solutions to be spread on the support, perform different concentration tests.</br>
+
                  <U>Materials:</U></br>
−
The cellulose being an integral part of the patch, we need to determine different solubility levels in order to constitute the correct mix for forming a solid support.</br></br>
+
&bull; Cellulose Avicell (Sigma-Aldrich)</br>
−
<U>Manipulation:</U></br>
+
&bull; BSA Protein</br>
−
&bull; cellulose solution in water and ethanol</br>
+
&bull; Rocker</br>
−
&bull; cellulose used = carboxymethyl-cellulose (CMC)</br>
+
&bull; Centrifuge</br>
−
&bull; Total volume = 40 ml solutions -> 40 g precisely (final weight)</br></br>
+
&bull; Laemmli 2X</br>
 +
&bull; Buffer A (50mM Tris, 150mM of NaCl)</br>
 +
&bull; Heating table</br>
 +
&bull; SDS-Page gel and cuve</br>
 +
&bull; Protein ruler</br>
 +
&bull; Microbiology equipment</br></br>
  
−
<U>Cellulose solutions in water :</U></br>
+
                  <U>Method:</U></br>
 +
1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br>
 +
2.In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 &#181;L of BSA solution and 10mg of Avicell.</br>
 +
3.Let rocking 1h at room temperature</br>
 +
4.Centrifuge 2min at 13.000 rpm</br>
 +
5.Take 1mL of the supernatent and store it in a 2mL eppendorf</br>
 +
6.Resuspend the pellet in 1mL of Buffer A</br>
 +
7.Vortex and let rocking during 2min at RT</br>
 +
8.Centrifuge 2min at 13.000rpm</br>
 +
9.Collect the supernatent and pool it with the first taken</br>
 +
10.Resuspend the pellet in 800 &#181;L of Laemmli 2X</br>
 +
11.Denaturate the protein by heating them at 95°C during 5min</br>
 +
12.Centrifuge 2min at 13.000rpm</br>
 +
13.Depose 20 &#181;L of the supernatent</br>
 +
14.For the other samples: mix 10 &#181;L of Laemmli 2X with 10 &#181;L of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br>
  
 +
<U>Deposit table on the gel:</U></br>
 +
Protein ruler</br>
 +
///</br>
 +
Pellet</br>
 +
///</br>
 +
Supernatant</br>
 +
///</br>
 +
Prep2</br>
 +
///</br>
 +
BSA</br></br>
  
−
  <table>
+
Start at 10:15AM</br></br>
−
                    <thead>
+
15. Wash the gel three times with distilled water during 5min.</br>
−
                        <tr>
+
16. Color the gel with Coomassie Blue diluted 1/5 during 30min.</br>
−
                            <th>Solution</th>
+
17. Wash with distilled water for 5min then let wash 15min.</br></br>
−
                            <th>% weight referred</th>
+
</p>
−
                            <th>m (cellulose) referred (g)</th>
+
−
                            <th>m final 5 (g)</th>
+
−
                            <th>m water added</th>
+
−
 
+
−
                        </tr>
+
−
                  </thead>
+
−
                    <tbody>
+
−
                          <tr>
+
−
                            <td><strong><p>(1)</p></strong></td>
+
−
                            <td>1.25</td>
+
−
                            <td>0.5</td>
+
−
                            <td>0.548</td>
+
−
                            <td>40.162</td>
+
−
                          </tr>
+
−
                          <tr>
+
−
                            <td><strong><p>(2)</p></strong></td>
+
−
                            <td>2</td>
+
−
                            <td>0.8</td>
+
−
                            <td>0.818</td>
+
−
                            <td>39.541</td>
+
−
</tr>
+
−
                          <tr>
+
−
                            <td><strong><p>(3)</p></strong></td>
+
−
                            <td>3</td>
+
−
                            <td>1.2</td>
+
−
                            <td>1.216</td>
+
−
                            <td>40.401</td>
+
−
                          </tr>
+
−
                          <tr>
+
−
                            <td><strong><p>(4)</p></strong></td>
+
−
                            <td>5</td>
+
−
                            <td>2</td>
+
−
                            <td>2.052</td>
+
−
                            <td>38.305</td>
+
−
                          </tr>
+
−
                          <tr>
+
−
                            <td><strong><p>(5)</p></strong></td>
+
−
                            <td>10</td>
+
−
                            <td>4</td>
+
−
                            <td>4.029</td>
+
−
                            <td>36.192</td>
+
−
                          </tr>
+
−
                          <tr>
+
−
                            <td><strong><p>(6)</p></strong></td>
+
−
                            <td>15</td>
+
−
                            <td>6</td>
+
−
                            <td>6.099</td>
+
−
                            <td>35.179</td>
+
−
                          </tr>
+
−
                          <tr>
+
−
                            <td><strong><p>(7)</p></strong></td>
+
−
                            <td>20</td>
+
−
                            <td>8</td>
+
−
                            <td>/</td>
+
−
                            <td>/</td>
+
−
</tr>
+
−
 
+
−
                      </tbody>
+
−
                  </table>
+
−
                  <center>Table 1: Masses</center></br></br></br>
+
−
 
+
−
 
+
−
N.B:  Given the excessive viscosity of the 15% to 20% solution was not prepared.</br></br>

Solution of cellulose in EtOH: </br>
+
−
2% by weight referred, 0.8 g referred cellulose.</br>
&#8594; 0.828 g of cellulose weighed; final weight</br>
<U>Total:</U> 35.317 g -> 2.3%</br></br>

These solutions were prepared in a 50 ml glass beaker covered with plastic film and left under magnetic stirring overnight at room temperature.</br>
In the different EtOH solutions &#8594; pure EtOH does not dissolve: solutions dissolve well for below 10% but are not homogeneous when content reach15%.</br></br>
+
−
 
+
−
<U>Conclusion/ Results:</U></br>
+
−
 
+
−
EtOH does not dissolve the cellulose, H20 is the best solvent.</br>
&#8594; The solutions below 10% are homogeneous,  but above 15% they  are not (it is too viscous).</br>
+
−
</p>
+
 
</div>
 
</div>
  
 +
<div id="J2"><h2><B> August 25th, 2016:</B> </h2></br></br></div>
 +
                <h3> <strong>Cellulose binding test </strong></h3>
 +
    <div class= "text1">
 +
<p>
 +
                <U> Aim:</U> This step checks the ability of our protein to bind cellulose.
 +
Moreover, this step will confirm the good design of the protein.</br></br>
  
 +
                  <U>Materials:</U></br>
 +
&bull; Cellulose Avicell (Sigma-Aldrich)</br>
 +
&bull; Cellulose Sigmacell (Sigma-Aldrich)</br>
 +
&bull; Carboxymethylcellulose (Sigma-Aldrich)</br>
 +
&bull; BSA Protein</br>
 +
&bull; Rocker</br>
 +
&bull; Centrifuge</br>
 +
&bull; Laemmli 2X</br>
 +
&bull; Buffer A (50mM Tris, 150mM of NaCl)</br>
 +
&bull; Heating table</br>
 +
&bull; SDS-Page gel and cuve</br>
 +
&bull; Protein ruler</br>
 +
&bull; Microbiology equipment</br></br>
  
−
<div>
+
<U>Method:</U></br> This protocol is done three times with the different cellulose.</br>
−
<p></br></br></br></p></div>
+
1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br>
 +
2.In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 &#181;L of BSA solution and 10mg of Avicell.</br>
 +
3.Let rocking 1h at room temperature</br>
 +
4.Centrifuge 2min at 13.000 rpm</br>
 +
5.Take 1mL of the supernatent and store it in a 2mL eppendorf</br>
 +
6.Resuspend the pellet in 1mL of Buffer A</br>
 +
7.Vortex and let rocking during 2min at RT</br>
 +
8.Centrifuge 2min at 13.000rpm</br>
 +
9.Collect the supernatent and pool it with the first taken</br>
 +
10.Resuspend the pellet in 800 &#181;L of Laemmli 2X</br>
 +
11.Denaturate the protein by heating them at 95°C during 5min</br>
 +
12.Centrifuge 2min at 13.000rpm</br>
 +
13.Depose 20 &#181;L of the supernatent</br>
 +
14.For the other samples: mix 10 &#181;L of Laemmli 2X with 10 &#181;L of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br>
  
−
<h2><B>May 26th, 2016:</B> </h2>
+
<U>Deposit table on the gel:</U></br>
−
<h3>Spreading test solutions of cellulose  on different supports: </h3>
+
Protein ruler</br>
−
+
///</br>
−
<div id= "J2">
+
Pellet (BSA + protein + Sigmacell)</br>
−
<p>
+
Supernatant (BSA + protein + Sigmacell)</br>
−
<U> Aim:</U> </br>Determine the solid support on which the spreading of the cellulose solution is optimal in view of evaporation of the solvent.</br>
The cellulose based patch needs to be created out of the dissolved cellulose solutions; in order to do that the solvent needs to be evaporated by spreading the mix over a solid support.</br></br>
+
Pellet (BSA + Sigmacell)</br>
 +
Supernatant (BSA + Sigmacell)</br></br>
  
−
<U>Principle:</U> </br>Spreading on media, qualitative evaluation of substrate wetting, and evaporation.</br></br>
+
Pellet (BSA + protein + Avicell)</br>
 +
Supernatant (BSA + protein + Avicell)</br></br>
  
−
<U>Manipulation:</U> </br>Spread with a flat spatula and qualitative assessment of the dampening (flexibility of the constituted solid surface).</br></br>
+
Pellet (BSA + protein + CMC)</br>
 +
Supernatant (BSA + protein + CMC)</br>
 +
Pellet (BSA + CMC)</br>
 +
Supernatant (BSA + CMC)</br></br>
  
−
<h4>With 3% solution:</h4>
 
−
&bull; with glass: very little grip, grainy materail that are not separated  by spreading.</br>
 
−
&bull; with paper filter: moderately satisfactory result, good flow, good adhesion but the filter paper is soaked.</br>
 
−
&bull; with aluminim: it spreads well in the beginning but dry very quickly and becomes granular in a few seconds.</br>
 
−
&bull; with a PVC film: same as glass with less ease of spreading.</br></br>
 
  
−
<h4>With 1.25% solution:</h4>
+
Start at 11:10AM at 130V. End at 12:20AM.</br></br>
−
&bull; with aluminum: a result slightly better but with holes that are always formed, the material dries.</br>
+
15. Wash the gel three times with distilled water during 5min.</br>
−
&bull; with the filter paper: a result a little better, good flow.</br>
+
16. Color the gel with Coomassie Blue diluted 1/5 during 30min.</br>
−
&bull; with PVC: same.</br>
+
17. Wash with distilled water for 5min then let wash 15min.</br></br>
−
&bull; with glasses: same.</br></br>
+
                   
 +
</p>
 +
</div>
  
−
<U>Problem: </U></br>The glass and aluminum does not give quite satisfactory results.</br></br>

        <h4>With the 5% solution:</h4></br>
+
<div id= "J3">
−
Same with the glass and aluminum &#8594; easier spreading at the beginning then more difficult because of the surface tension, but with a slower reaction because of the higher viscosity. 5% on normal paper: good texture after wetting, good flow, low drying.</br></br>

The most effective materials are aluminum and the filter paper.</br></br>
+
<p></br></br></br> </p>
 +
</div>
  
−
Plate making test: spread then put in an oven at 50 °C:</br></br>

Test plates:</br></br>
+
<div class= "text1"><h2><B> August 29th, 2016:</B></h2></div></br></br>
 +
<h3> <strong>Cellulose binding test</strong> </h3>
  
−
<img src="" width="75%"  alt="image"/></img></br></br>
+
    <div class= "text1">
 +
  <p>
  
−
The solutions were spread through a calibrated wiper system to the surface is as flat as possible a layer.</br>
The plates are placed in an incubator 50 °C for 20 hours.</br></br>

Another test has been performed: on a glass plate in a 10% solution that has been deposited on cotton and the plate is placed in an oven.</br></br>
+
              <U> Aim:</U> This step checks the ability of our protein to bind cellulose.
 +
Moreover, this step will confirm the good design of the protein.</br></br>
  
−
<U>Results of drying in the oven:</U></br>
Cellulose deposits are invisible on paper. Even scratching does not cause the cellulose grain to coming off.</br>
On aluminum, 10% solution of cellulose gave some stuck grains that couldn't be removed by scratching.</br></br>

<U>Conclusion:</U> </br>The cellulose concentration is too low.</br></br>

+
              <U>Materials:</U></br>
−
The deposit on cotton gave an unexpected result: the solution solidified into a flexible film. The surface is uneven and therefore of  little use for our patch. To try to obtain a homogeneous film, several other experiments are conducted.</br></br>
&#8594; The 10% solution is placed between two glass plates and then placed in an oven</br>
&#8594; Another plating on aluminum is done. To avoid the wetting phenomenon which makes the convex surface, the walls were made by inserting the foil in a rectangular mold. Two tests are performed:</br>
+
−
&bull; The foil is filled with a 10% solution</br>
+
−
&bull; The foil is filled with a 10% solution and some cotton fibers are added over the paper. If the result is a film, the planar surface would be one that is below (where there is no cotton fibers). These plates are dried in open air for 5 days as an oven is unavailable.
+
−
</br></br>
+
  
−
<h4>Testing a mixture of solvent: water-ethanol.</h4></br>
<U>Aim :</U></br> Dissolving cellulose in a water-ethanol mixture to use the fact that these two species form  an azeotropic mixture.</br></br>

<h4>Evaporating the mixture, ethanol will leave more easily than water, which will increase the cellulose concentration in water and bring it to solidify.</h4>
</br>
</h4>One must test x % EtOH that is less than 90%, such that more ethanol will evaporate, thus forcing the cellulose to solidify more. Two solutions are prepared:</h4></br></br>
+
  &bull; Cellulose Avicell (Sigma-Aldrich)</br>
−
&bull; xetOH =  50%</br>
+
  &bull; BSA Protein</br>
−
&bull; xetOH = 70%</br></br>
+
  &bull; Rocker</br>
 +
  &bull; Centrifuge</br>
 +
  &bull; Laemmli 2X</br>
 +
  &bull; Buffer A (50mM Tris, 150mM of NaCl)</br>
 +
  &bull; Heating table</br>
 +
  &bull; SDS-Page gel and cuve</br>
 +
  &bull; Protein ruler</br>
 +
  &bull; Microbiology equipment</br></br>
  
−
Unfortunately, these solutions do not dissolve cellulose. So the deposit appears to be too heterogeneous for sedimentation; it would result in basically unequal amounts of cellulose.</br></br>

<U>Conclusion: </U></br>the water-ethanol mixture behaves like the ethanol solution.</br>
+
<U>Method:</U></br>
−
</p>
+
1.Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br>
−
</div>
+
2.In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 100 &#181;L of BSA solution and 10mg of Avicell.</br>
 +
3.Let rocking 1h at room temperature</br>
 +
4.Centrifuge 2min at 13.000 rpm</br>
 +
5.Take 1mL of the supernatent and store it in a 2mL eppendorf</br>
 +
6.Resuspend the pellet in 1mL of Buffer A</br>
 +
7.Vortex and let rocking during 2min at RT</br>
 +
8.Centrifuge 2min at 13.000rpm</br>
 +
9.Collect the supernatent and pool it with the first taken</br>
 +
10.Resuspend the pellet in 800 &#181;L of Laemmli 2X</br>
 +
11.Denaturate the protein by heating them at 95°C during 5min</br>
 +
12.Centrifuge 2min at 13.000rpm</br>
 +
13.Depose 20 &#181;L of the supernatent</br>
 +
14.For the other samples: mix 10 &#181;L of Laemmli 2X with 10 &#181;L of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br>
  
−
<div class= "text1">
 
−
<p></br></br></br> </p>
 
−
</div>
 
  
−
<h2><B>May 27th, 2016:</B> </h2>
+
<U>Deposit table on the gel:</U></br>
−
<h3>Compression of the powder or a powder-water mixture to make the patch. </h3>
+
Protein ruler</br>
−
<div id= "J3">
+
///</br>
−
  <p>
+
Pellet</br>
−
<U> Aim:</U></br>
+
///</br>
Supernatant 1</br>
−
Produce a patch with pure cellulose powder to avoid the problem of too low concentration. After the difficulties with the water/EtOH_Cellulose mixtures, we decided to use pure cellulose in a compressed form.</br></br>
+
///</br>
 +
Supernatant 2</br>
 +
///</br>
 +
Supernatant 3</br> 
 +
///</br>
 +
Prep2</br>
 +
///</br>
BSA</br></br>
  
−
<U> Principle:</U> </br>
+
15. Wash the gel three times with distilled water during 5min.</br>
−
Compress the powder to assess the surface produced by such method. The shape of the patch is given by a mold used in the pressor compartment.</br>
+
16. Color the gel with Coomassie Blue diluted 1/5 during 30min.</br>
−
The powder is first compressed into a rectangular mold 1 mm thick at 200 bar and at room temperature. The result is quite satisfactory: a friable solid is obtained.</br></br>

To harden the materiel obtained, the same experiment is conducted by adding a few drops of water on top. After compression, the powder was solidified in part and a few grainy bits remained. It is therefore necessary to mix water and the powder before compressing. A water-cellulose mixture is then prepared: 3.42 g of cellulose and 3 g of water.</br></br>
+
17. Wash with distilled water for 5min then let wash 15min.</br></br>
−
 
+
</p>
−
It gives a mixture of 53% cellulose. It has a paste like texture.</br></br>

The compression plates have been heated beyond our control. We proceeded with cutting the shapes are by lack of time.</br>
The only powder is compressed into a mold in the form of 0.5 mm thick test piece. plate temperature is 90 °C. The result is the same, a brittle solid.</br></br>

The mixture at 53 °C cellulose is then compressed at 80 °C in the same mold. A flexible and resistant film is obtained. It is a bit transparent.</br></br>

The patches obtained are then immersed in water in order to simulate for the reaction of the patch to the incorporation of proteins and to the silification.</br></br>
+
−
 
+
−
We observed that after 1 minute 45 s, the patch from the cellulose-water mixture softens but doesn't disaggregate. It can be recovered in a solid form.</br></br>

After 2 min, the patch from the cellulose only (without water)  powder curves but remains strong. </br></br>
+
−
 
+
−
The patches are then allowed to dry in the open area.</br></br>

<U>Conclusion:</U> </br> This method is suitable for manufacturing. It remains to determine how to incorporate protein and silica.</br>
+
−
</p>
+
 
</div>
 
</div>
  
−
<div class="text1">
+
<div id="J4">
 
<p></br></br></br> </p>
 
<p></br></br></br> </p>
 
</div>
 
</div>
  
−
<h2><B>May 28th, 2016:</B></h2>
+
<div class="text1"><h2><B> September 12th, 2016: </B></h2></div></br></br>
−
<h3>Improvements </h3>
+
<h3><strong> Cellulose binding test </strong></h3>
  
−
<div id="J4">
+
<div class="text1">
 
<p>
 
<p>
 +
     
 +
              <U> Aim:</U> This step checks the ability of our protein to bind cellulose.
 +
Moreover, this step will confirm the good design of the protein.</br></br>
  
−
<U>  Aim:</U> </br>  Improve cellulose patch and drying results. We have pursued the testing of the patches for mechanical properties and for resistance to water in order to mimic the conditions that would be faced in the presence of protein solutions.</br></br>
 
  
−


<U>Results of drying after immersion patches of <i>May 27, 2016</i>:</U>
+
              <U>Materials:</U></br>
−
Both patches have hardened to give a patch that holds itself (good mechanical properties).</br>
One obtains a similar result to a higher concentration of water patch.</br>
&#8594; We keep the mechanical strength after immersion and drying. This result is similar to the texture we are looking for our patch.</br></br>

<h4>Paper cups drying aluminum </h4>
+
&bull; Cellulose Avicell (Sigma-Aldrich)</br>
−
<U>Result:</U></br>
+
&bull; BSA Protein</br>
−
&bull; Heterogeneous deposit and fines in the mold without cotton fibers.</br>
+
&bull; Rocker</br>
−
&bull; The one with the cotton fibers has given a rigid film thus a fixed cotton-cellulose composite and shows  added mechanical strength.</br>
+
&bull; Centrifuge</br>
 +
&bull; Laemmli 2X</br>
 +
&bull; Buffer A (50mM Tris, 150mM of NaCl)</br>
 +
&bull; Heating table</br>
 +
&bull; SDS-Page gel and cuve</br>
 +
&bull; Protein ruler</br>
 +
&bull; Microbiology equipment</br></br>
  
−
</p>
+
<U>Method:</U></br>
−
</div>
+
1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br>
 +
2. In a 1.5mL eppendorf, put 1mL of protein solution (previously purified thanks to FPLC), 10 &#181;L of BSA solution and 10mg of Avicell.</br>
 +
3. Let rocking 1h at room temperature</br>
 +
4. Centrifuge 2min at 13.000 rpm</br>
 +
5. Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br>
 +
6. Resuspend the pellet in 1mL of Buffer A</br>
 +
7. Vortex and let rocking during 2min at RT</br>
 +
8. Centrifuge 2min at 13.000rpm</br>
 +
9. Collect the supernatent in a clean eppendorf (Supernatant 2)</br>
 +
10. Resuspend the pellet in 1mL of Buffer A</br>
 +
11. Vortex and let rocking during 2min at RT</br>
 +
12. Centrifuge 2min at 13.000rpm</br>
 +
13. Collect the supernatent in a clean eppendorf (Supernatant 3)</br>
 +
14. Resuspend the pellet in 200 &#181;L of Laemmli 2X</br>
 +
15. Denaturate the protein by heating them at 95°C during 5min</br>
 +
16. Centrifuge 2min at 13.000rpm</br>
 +
17. Depose 20 &#181;L of each supernatent</br>
 +
18. For the other samples: mix 10 &#181;L of Laemmli 2X with 10 &#181;L of protein (BSA alone, Prep2 after dialysis). Denaturate them 5min at 95°C</br></br>
  
−
<div class="text1">
+
<U>Deposit table on the gel:</U></br>
−
<p></br></br></br> </p>
+
Protein ruler</br>
−
</div>
+
///</br>
 +
Pellet</br>
 +
///</br>
Supernatant 1</br>
 +
///</br>
 +
Supernatant 2</br>
 +
///</br>
 +
Supernatant 3</br> 
 +
///</br>
 +
Prep2</br>
 +
///</br>
BSA</br></br>
  
−
<h2><B>June 3th, 2016:</B></h2>
+
19. Wash the gel three times with distilled water during 5min.</br>
−
<h3> Preparation of other patches:</h3>
+
20. Color the gel with Coomassie Blue diluted 1/5 during 30min.</br>
−
<div id="J5">
+
21. Wash with distilled water for 5min then let wash 15min.</br></br>
−
<p>
+
−
<U> Aim:</U> </br>  Produce patches with the cellulose powder by testing other cellulose concentrations, and other temperatures. We would like to explore other conditions for formong the patches, thereore we plan other experiments including other ratios of water:cellulose, pressures and temperatures. </br> </br>
+
−
<U> Principle:</U> </br> Compress the powder (with water optionally) with a press for discharging air in the cellulose powder, or for expelling excess water. The shape of the patch is given by a mold. </br> </br>

+
  
−
<U> Manipulation:</U> </br>  
&bull; Preparation of a 75% paste, composed of 4.5 g of cellulose + 1.5 g of water &#8594; Paste A </br>
&bull; Preparation of another pulp 75%, 4.5 g cellulose + 1.5 g of water &#8594; Paste B </br>
&bull; Preparation of a pulp 65%, 3.9 g cellulose + 2.1 g of water &#8594; Paste C </br> </br>
+
</p>
−
&bull; Compression of the Paste A to 200 bar at room temperature (2 min). We obtained an opaque patch, some areas have dried, there is a good mechanical strength, not too brittle. </br> </br>
+
−
&bull; Compression of the Paste B to 200 bars at 70 ° C (2 min). We obtained a patch with interesting properties: it has intermediate mechanical properties, smooth, more rigid than that at 65% and 53%; however, it is somewhat brittle. </br> </br>
+
−
&bull; Compression of the Paste C to 200 bars at 70 ° C (2 min). A translucent patch is obtained, flexible, almost identical to Paste B. </br> </br> </br>
+
−
 
+
−
<U> Conclusion:</U> </br>  The pulp at 75% cellulose seems to have given the most interesting result for its mechanical properties and its apparent homogeneity before and after immersion / drying. The temperature of 70 °C seems well suited to the specifications of the patch. </br>
+
−
</p>
+
 
</div>
 
</div>
  
  
−
<div class="text1">
+
<div id="J5">
 
<p></br></br></br> </p>
 
<p></br></br></br> </p>
 
</div>
 
</div>
−
 
+
<div class="text1"><h2><B> September 14th, 2016: </B></h2></div></br></br>
−
 
+
<h3> <strong>Cellulose binding test</strong> </h3>
−
<h2><B>July 7th, 2016: </B></h2>
+
<div class="text1">
−
<h3> Compression of new patches with Kapton tape: </h3>
+
−
<div id="J6">
+
 
<p>
 
<p>
−
<U> Aim: </U> </br> Make new patches bu using Kapton tape (adhesive coating, Dupont) to strengthen them. Test different concentrations, temperatures, and types of cellulose. (+ influence of a protein on compression). We need a solid surface on which to lay our cellulose patching fabrication. After testing paper, cotton, aluminum and plastic, we will test Kapton, which is used as a tape in spacecraft industry. </br> </br>
 
−
 
−
<U> Principle:  </U> </br> Compression of microcrystalline cellulose powder with a press to evacuate air. The shape of the patch is given by a mold. </br> </br>
 
−
<img src="" width="75%"  alt="image"/></img>
 
−
<U> Manipulation:</U> </br>
 
−
&bull; Preparation of two 100% cellulose patches to determine the actual weight of powder required to make a patch (2 types of cellulose: Avicel and Sigmacell- 50 µm crystal MCC) </br> </br>
 
−
 
−
<strong>Patch n°:</strong> </br> </br>
 
−
1) 100% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
 
−
2) 100% Avicel, 200 bars, 2 min, RT, Kapton tape </br> </br>
 
−
 
−
<i>N.B.: 3 g of powder are required to make a patch (2 g display) </i></br>
 
−
&bull; Preparation of two patches (one with a protein and another without) to determine whether a protein has an influence on compression. </br>
 
−
Solution of protein: 3 mg of BSA (Bovine Serum Albumin) in 1 ml of distilled water. </br> </br>
 
−
 
−
<strong>2 patches:</strong> </br> </br>
 
−
3) 50% (1 g cellulose + 1 ml BSA solution at 3 mg/ml), Sigmacell, 200 bars, 2 min, RT, adhesive Kapton </br>
 
−
4) 50% (1 g cellulose + 1 ml water), Sigmacell, 200 bars, RT, adhesive Kapton, 2 min. </br>
 
−
 
−
&#8594; The presence of protein doesn’t change anything anything to the mechanical properties of the patch. </br>
 
−
We can therefore proceed with the experiment the point when well will need to add our protein to the patch.
 
−
</br> </br>
 
−
<U> Aim: </U> </br> We need to start again this experiment since the protein wasn’t in a buffer (Tris, NaCl)&#8594;hydrophobic parts aggregate &#8594; which may lead to phase separation. We neede to test this environment for the protein, as unlie BSA which behave moderately well in distilled water, we have to anticipate that or protein construct which will be a fusion protein, might require a buffer to keep it in a stable state. </br> </br>
 
−
 
−
<U>Preparation of several patches to test concentration: </U></br>
 
−
 
−
<table>
 
−
                    <thead>
 
−
                        <tr>
 
−
                            <th>w/w %</th>
 
−
                            <th>Cellulose weight (g)</th>
 
−
                            <th>Water weight (g)</th>
 
−
                        </tr>
 
−
                  </thead>
 
−
                    <tbody>
 
−
                          <tr>
 
−
                            <td><strong><p>60</p></strong></td>
 
−
                            <td>1.8</td>
 
−
                            <td>1.2</td>
 
−
                          </tr>
 
−
                          <tr>
 
−
                            <td><strong><p>70</p></strong></td>
 
−
                            <td>2.1</td>
 
−
                            <td>0.9</td>
 
−
</tr>
 
−
                          <tr>
 
−
                            <td><strong><p>80</p></strong></td>
 
−
                            <td>2.4</td>
 
−
                            <td>0.6</td>
 
−
                          </tr>
 
−
                          <tr>
 
−
                            <td><strong><p>90</p></strong></td>
 
−
                            <td>2.7</td>
 
−
                            <td>0.3</td>
 
−
                          </tr>
 
−
                      </tbody>
 
−
                  </table>
 
−
                  <center>Table 2: Quantities</center></br></br></br>
 
−
 
−
 
  
−
<strong>Patch n°:</strong></br> </br>
+
              <U> Aim:</U> This step checks the ability of our protein to bind cellulose.</br>
−
5) 60% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
+
Moreover, this step will confirm the good design of the protein.</br>
−
6) 70% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
+
−
7) 80% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
+
−
8) 90% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
+
−
9) 50% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
+
−
10) 60% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
+
−
11) 70% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
+
−
12) 80% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br>
+
−
13) 90% Sigmacell, 200 bars, 2 min, RT, Kapton tape </br> </br>
+
  
−
&#8594; The best concentration appears to be 70% w/w for homogeneity, mechanical resistance…etc </br> </br>
+
              <U>Materials:</U></br>
−
&bull; Below 70%, the patch contains too much water on cellulose doesn’t stick to the tape </br>
+
&bull; Cellulose Avicell (Sigma-Aldrich)</br>
−
&bull; Above 70%, the patch is too dry, it crumbles </br>
+
&bull; BSA Protein</br>
−
&bull; Test with 2 layers of Kapton tape: </br> </br>
+
&bull; Rocker</br>
 +
&bull; Centrifuge</br>
 +
&bull; Laemmli 2X</br>
 +
&bull; Buffer A (50mM Tris, 150mM of NaCl)</br>
 +
&bull; Heating table</br>
 +
&bull; SDS-Page gel and cuve</br>
 +
&bull; Protein ruler</br>
 +
&bull; Microbiology equipment</br></br>
  
  
−
<img src="" width="75%"  alt="image"/></img>
+
<U>Method:</U></br>
−
14) 2 x (200 bars, 2 min, RT) 2 Kapton layers, 70% </br>
+
1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br>
−
&#8594; good mechanical properties, but it still crumbles between the 2 Kapton layers silica required! </br> </br>
+
2. In a 1.5mL eppendorf, follow the next volume table:</br>
−
&bull; Test with 2 layers of Kapton tapes at different temperatures </br> </br>
+
<table>
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Tube</th>
 +
                            <th>24kDa - sample 1</th>
 +
                            <th>24kDa - sample 2</th>
 +
                            <th>24kDa - sample 3</th>
 +
                            <th>40kDa - sample 1</th>
 +
                            <th>40kDa - sample 2</th>
 +
                            <th>40kDa - sample 3</th>
 +
                        </tr>
 +
                  </thead>
 +
                    <tbody>
 +
                          <tr>
 +
                            <td><strong><p>Protein solution</p></strong></td>
 +
                            <td>500 &#181;L </td>
 +
                              <td>250 &#181;L </td>
 +
                            <td>500 &#181;L </td>
 +
                            <td>500 &#181;L </td>
 +
                            <td>250 &#181;L </td>
 +
                            <td>500 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>BSA</p></strong></td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Avicell</p></strong></td>
 +
                            <td>10 mg </td>
 +
                            <td>10 mg</td>
 +
                            <td>10 mg </td>
 +
                            <td>10 mg </td>
 +
                            <td>10 mg </td>
 +
                            <td>10 mg </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Buffer A</p></strong></td>
 +
                            <td>500 &#181;L </td>
 +
                              <td>750 &#181;L </td>
 +
                            <td>500 &#181;L </td>
 +
                            <td>500 &#181;L </td>
 +
                            <td>750 &#181;L </td>
 +
                            <td>500 &#181;L </td>
 +
                          </tr>
 +
                                              </tbody>
 +
                  </table>
 +
                  <center>Volumes</center></br></br></br>
  
−
15) 2 x (200 bars, 2 min, 50°C) 2 Kapton layers, 70% </br>
 
−
16) 2 x (200 bars, 2 min, 80°C) 2 Kapton layers, 70% </br> </br>
 
  
−
&#8594;80°C: still crumbling, white (and not transparent like the one with carboxymethyl cellulose)&#8594;water evaporated
+
3. Let rocking 2h at room temperature</br>
−
</br>
+
4. Centrifuge 2min at 13.000 rpm</br>
−
&#8594;50°C: good, still water, homogeneous. </br> </br></br>
+
5. Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br>
 +
6. Resuspend the pellet in 1mL of Buffer A</br>
 +
7. Vortex and let rocking during 2min at RT</br>
 +
8. Centrifuge 2min at 13.000rpm</br>
 +
9. Collect the supernatent in a clean eppendorf (Supernatant 2)</br>
 +
10. Resuspend the pellet in 200 &#181;L of Laemmli 2X</br>
 +
11. Denaturate the protein by heating them at 95°C during 5min</br>
 +
12. Centrifuge 2min at 13.000rpm</br>
 +
13. Depose 40 &#181;L of each supernatent</br>
 +
14. For the other samples: mix 20 &#181;L of Laemmli 2X with 20 &#181;L of protein (BSA alone, protein solution alone). Denaturate them 5min at 95°C</br></br>
  
−
<U>Conclusion:</U> </br> The best patch seems to be the double layered, 50°C, 70% w/w one. </br>
+
<U>Deposit table on the gel:</U></br>
 +
BSA (x2)</br>
 +
///</br>
Supernatant 1 (x6)</br>
 +
///</br>
 +
Supernatant 2 (x6)</br>
 +
///</br>
 +
Protein 24kDa</br>
 +
///</br>
 +
Protein 40kDa</br>
 +
///</br>
Pellet (x6)</br></br>
  
−
However, we must find how to get the properties and the flexibility of carboxymethyl cellulose (CMC) 65°C patch (200 bars, 80°C) </br> </br>
+
15. Wash the gel three times with distilled water during 5min.</br>
 +
16. Color the gel with Coomassie Blue diluted 1/5 during 30min.</br>
 +
17. Wash with distilled water for 5min then let wash 15min.</br></br>
  
−
Picture 1-Sigmacell cellulose patches </br> </br>
+
</p>
−
Picture 2- Avicel cellulose patches </br> </br>
+
−
&bull; Avicel patches are better (crumble less, better cohesion) </br>
+
−
&bull; The more water there is, the less Kapton tape is efficient to stick cellulose powder. </br> </br>
+
−
 
+
−
Picture 3- Kapton double layered patches </br> </br>
+
−
 
+
−
&#8594; flexible </br>
+
−
&#8594;transparent </br>
+
−
&#8594;homogeneous </br> </br>
+
−
</p>
+
 
       </div>
 
       </div>
  
−
<div class= "text1">
+
<div id="J6">
 
<p></br></br></br> </p>
 
<p></br></br></br> </p>
 
</div>
 
</div>
−
<h2><B>July 22th, 2016: </B></h2>
+
              <div class= "text1"><h2><B> September 29th, 2016: </B></h2></div></br></br>
−
<h3> Test of the feasibility of the detection device with Coomasie blue </h3>
+
<h3> <strong>Cellulose binding test </strong></h3>
 
 
  
−
<div id="J7">
+
<div class= "text1">
 
<p>
 
<p>
−
<U> Aim: </U> </br>See if it is possible to use Coomasie blue to reveal the presence of the viral protein. </br> Our aim in this experiment is to evaluate the amount of protein retained on the cellulose layer. We will use the capacity of Coomassie blue to stain proteins (lysine, arginine) in order to estimate the amount bound. </br> </br>
 
  
−
<U> Principle: </U> </br>Pour Coomasie blue on the patch and wash it with water. </br> </br>
+
              <U> Aim:</U> This step checks the ability of our protein to bind cellulose.</br>
 +
Moreover, this step will confirm the good design of the protein purified on the 26/09.</br></br>
  
−
<U> Experiment:</U> </br>
+
              <U>Materials:</U></br>
−
&bull; The Coomassie blue was poured on the 65% carboxymethyl cellulose (CMC) and then, the patch was washed. </br>
+
&bull; Cellulose Avicell (Sigma-Aldrich)</br>
−
&bull; Washing the patch during around 15 seconds was enough to significantly remove the color. </br>
+
&bull; BSA Protein</br>
−
&bull; The same experiment is carried out on the patches 12, 11 and 9
+
&bull; Rocker</br>
−
Washing the patch during several seconds is not enough to remove the color
+
&bull; Centrifuge</br>
−
They are immersed in water for decoloration during 30 min. </br> </br>
+
&bull; Laemmli 2X</br>
−
We need to improve this method of estimation of the amount of protein bound. One other method is to soak the patch in a solution of known protein concentration, followed by the estimation of the amount of protein left after the patch has been removed. We plan to use the Bradford assay, using a standard curve based on BSA fo rthis experiment. </br> </br>
+
&bull; Buffer A (50mM Tris, 150mM of NaCl)</br>
 +
&bull; Heating table</br>
 +
&bull; SDS-Page gel and cuve</br>
 +
&bull; Protein ruler</br>
 +
&bull; Microbiology equipment</br></br>
  
  
−
<h4>Compression of new patches (without Kapton tape)</h4></br>
+
<U>Method:</U></br>
−
<U> Aim:</U> </br>Make new patches without Kapton tape. Test different temperatures and types of cellulose. Obtain the same flexibility and homogeneity as with the CMC patch (65%, 80°C, 200 bars). </br> </br>
+
1. Mix BSA with Buffer A to reach a concentration of 10mg/mL of BSA (10mg of BSA in 1mL of Buffer A)</br>
 +
2. In a 1.5mL eppendorf, follow the next volume table:</br>
  
−
<U> Principle:</U> </br> Compression of microcrystalline cellulose and CMC powder with a press to evacuate air. The shape of the patch is given by a mould. </br> </br>
+
<table>
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Fractions</th>
 +
                            <th>14</th>
 +
                            <th>15</th>
 +
                            <th>16</th>
 +
                            <th>19</th>
 +
                            <th>20</th>
 +
                            <th>BSA control</th>
 +
                        </tr>
 +
                  </thead>
 +
                    <tbody>
 +
                          <tr>
 +
                            <td><strong><p>Concentration (ng/&#181;L )</p></strong></td>
 +
                            <td>782 &#181;L </td>
 +
                              <td>1086 &#181;L </td>
 +
                            <td>1157 &#181;L </td>
 +
                            <td>982 &#181;L </td>
 +
                            <td>980 &#181;L </td>
 +
                            <td>1000 &#181;L </td>
 +
                          </tr>                          <tr>
 +
                            <td><strong><p>Protein solution</p></strong></td>
 +
                            <td>300 &#181;L </td>
 +
                              <td>300 &#181;L </td>
 +
                            <td>300 &#181;L </td>
 +
                            <td>300 &#181;L </td>
 +
                            <td>300 &#181;L </td>
 +
                            <td>0 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>BSA</p></strong></td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>10 &#181;L </td>
 +
                            <td>300 &#181;L </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Avicell</p></strong></td>
 +
                            <td>10 mg </td>
 +
                            <td>10 mg</td>
 +
                            <td>10 mg </td>
 +
                            <td>10 mg </td>
 +
                            <td>10 mg </td>
 +
                            <td>10 mg </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td><strong><p>Buffer A</p></strong></td>
 +
                            <td>690 &#181;L </td>
 +
                            <td>690 &#181;L </td>
 +
                            <td>690 &#181;L </td>
 +
                            <td>690 &#181;L </td>
 +
                            <td>690 &#181;L </td>
 +
                            <td>690 &#181;L </td>
 +
                          </tr>
 +
                                              </tbody>
 +
                  </table>
 +
                  <center>Volumes</center></br></br></br>
  
  
−
<U> Experiments: </U> </br>CMC= carboxymethyl cellulose </br> </br>
+
3. Let rocking 2h at room temperature</br>
 +
4. Centrifuge 2min at 13.000 rpm</br>
 +
5. Take 1mL of the supernatent and store it in a 2mL eppendorf (Supernatant 1)</br>
 +
6. Resuspend the pellet in 1mL of Buffer A</br>
 +
7. Vortex and let rocking during 2min at RT</br>
 +
8. Centrifuge 2min at 13.000rpm</br>
 +
9. Collect the supernatent in a clean eppendorf (Supernatant 2)</br>
 +
10. Resuspend the pellet in 200 &#181;L of Laemmli 2X</br>
 +
11. Denaturate the protein by heating them at 95°C during 5min</br>
 +
12. Centrifuge 2min at 13.000rpm</br>
 +
13. Depose 40 &#181;L of each supernatent</br>
 +
14. For the other samples: mix 20 &#181;L of Laemmli 2X with 20 &#181;L of protein (BSA alone, protein solution alone). Denaturate them 5min at 95°C
 +
</br></br>
 +
<U>Deposit table on the gel:</U></br>
 +
Fraction 14</br>
 +
///</br>
Fraction 15</br>
 +
///</br>
 +
Fraction 16</br>
 +
///</br>
 +
Fraction 19</br>
 +
///</br>
 +
Fraction 20</br>
 +
///</br>
BSA control</br></br>
  
−
&bull; Preparation of the same CMC patch: (2.1 g cellulose / 0.9 g eau) 70%, RT, 2 min, bars, CMC (fig1) </br>
+
15. Wash the gel three times with distilled water during 5min.</br>
−
&#8594;The patch is flexible, homogeneous and transparent (we reached the expected result, this method is reproducible). This process works at room temperature  great! </br> </br>
+
16. Color the gel with Coomassie Blue diluted 1/5 during 30min.</br>
 +
17. Wash with distilled water for 5min then let wash 15min.</br></br>
  
−
&bull; Preparation of the same CMC patch with BSA/buffer </br>
+
</p>
−
- dissolution of 3 mg of BSA in 0.9 g of Tris/HCl buffer </br>
+
−
- mix with 2.1 g of CMC </br>
+
−
- compression: 200 bars, RT, 4min (with BSA) (fig 2) </br>
+
−
&#8594; We got a patch with a good flexibility but it’s not very homogeneous </br> </br>
+
−
 
+
−
&bull; Preparation of an Avial patch : 70% RT, 4min, 200 bars, Avial (fig3) </br>
+
−
&#8594; We got a white opaque, brittle patch, easily crumbled </br> </br>
+
−
 
+
−
&bull; We tried to increase the temperature of the press to get the same patch with Avial as the one with CMC. </br>
+
−
- Avial, 50°C, 4min 200 bars, 70% (fig5) </br>
+
−
- Avial, 80°C, 200 bars, 70% (fig 6) </br>
+
−
 
+
−
&#8594; At 80°C, the patch is more solid. </br>
+
−
&#8594; We do not succeed in making a flexible and transparent patch, neither at 50°CC nor at 80°C, with CMC. </br> </br>
+
−
 
+
−
The stiffness of the patches increases as they dry (true for all the patches). </br> </br>
+
−
 
+
−
 
+
−
Fig1/Fig2/Fig3/Fig4/Fig5/Fig6
+
−
 
+
−
Patches prepared on <i>July 22, 2016</i> </br>
+
−
</p>
+
−
</div>
+
−
 
+
−
+
−
<div class="text1">
+
−
<p></br></br></br> </p>
+
−
</div>
+
−
 
+
−
 
+
−
<h2><B>October 7th, 2016: </B></h2>
+
−
<h3> Test of binding protein-patch</h3>
+
−
<div id="J8">
+
−
<p>
+
−
<U>Aim:</U> </br> Prepare various patches with the silicated C2 protein in them, for immuno-assays and traction tests. </br> </br>
+
−
<U>Materials:</U> </br>
+
−
&bull; Avicell commercial cellulose </br>
+
−
&bull; Silicated C2 protein from previous weeks </br>
+
−
&bull; Scale and a press </br> </br>
+
−
<U>Protocol: </U> </br>see the patch compression protocol </br> </br>
+
−
<U>Experiment:</U> </br>
+
−
We prepared: </br>
+
−
&bull; a 100% Avicell patch </br>
+
−
&bull; a 100% CMC patch </br>
+
−
&bull; a 10mm diameter patch with 66%wt of Avicell: 50mg of silicated C2 protein + 100mg of Avicell </br>
+
−
&bull; a 10mm diameter patch with 66%wt of CMC: 50mg of silicated C2 protein + 100mg of CMC </br>
+
−
&bull; ten 3mm diameter patch with 50%wt of Avicell: 50mg of silicated C2 protein + 50mg of Avicell </br>
+
−
&bull; ten 3mm diameter patch with 66%wt of Avicell: 50mg of silicated C2 protein + 100mg of Avicell </br>
+
−
</p>
+
 
       </div>
 
       </div>
  
 
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Latest revision as of 20:33, 17 October 2016