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Revision as of 15:16, 27 September 2016

6x Laemmli SDS Sample Loading Buffer

Introduction

6x Protein Loading Buffer (Laemmli buffer) is used for the preparation of protein samples for SDS-poluacrylamide gel electrophoresis (SDS-PAGE). After the addition of the reducing agent β-mercaptoethanol (or DTT), the protein buffer will contain all of the necessary components for complete disruption of high-order protein structures. 6X Protein Loading Buffer is ideal because the protein sample prepared in 6X buffer will be more concentrated than protein sample prepared in 4X or 2X buffer (i.e. more protein and less loading buffer per well) SAFETY PRECAUTIONS SDS (safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets. PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back. HAZARDS: β-mercaptoethanol: Combustible Liquid, Toxic by inhalation., Toxic by ingestion, Toxic by skin absorption, Moderate skin irritant, Severe eye irritant

Materials

  • Reagents
    • 375mM Tris. HCL pH8.8
    • 9%SDS
    • 50% Glycerol
    • 0.03% Bromophenol blue
    • 14.7M β-mercaptoethanol
  • Equipment
    • Micropipetter + tips
    • Beaker
    • Waterbath
    • microcentrifuge
    • microcentrifuge tube

Procedure

  • Production of 100mL of stock
  1. Add 5.91 g Tris-HCl, 6 g SDS, 48 mL 100% glycerol, 9 mL 14.7 M 2-Mercaptoethanol, and 30 mg bromophenol blue. Bring to 100 mL with ddH2O.
A
B
1
Reagent Quantity
2
375mM Tris-HCl 5.91 g
3
9% SDS 6 g
4
50% Glycerol 48 mL
5
0.03% Bromophenol blue 30mg
6
MilliQ (ddH2O) top to bring total to 100mL
Table1
  1. Add 5.91 g Tris-HCl, 6 g SDS, 48 mL 100% glycerol, 9 mL 14.7 M 2-Mercaptoethanol, and 30 mg bromophenol blue. Bring to 100 mL with ddH2O.
  1. Mix well and dissolve any percipitates in sample loading buffer by incubating at 37oC. This solution can be used as stock solution.
  • Using 6X SDS Sample loading buffer
  1. Add 9μL β-mercaptoethanol to 91 μLSDS Protein Loading Buffer and mix well. Invert 10 times.
  1. Make a 1:5 dilution of SDS Protein Loading Buffer (containing the reducing agent) to protein sample.
  • For example add 1μL of buffer to 5μL of sample protein.
  1. Heat prepared protein sample at 100oC for 5 minutes.
  1. Breifly centrifuge heated sample and load into SDS polyacrylamide gel.
  • *****Note: B-mercaptoethanol rapidly oxidizes in protein loading buffer. Fresh 6X protein loading buffer shoudl be prepared every time!*****
  • Reference
  • http://www.wikiprotocols.org/protocols/formulation-of-6x-sds-sample-buffer/10090
  • http://www.morganvillesci.com/6X-SDS-Protein-Loading-Buffer-25-mL-LB0100.htm
  • Changelong
  • Created 5/17/2016