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Sample Preparation in Western Blot Assay

Contents

Lysis Buffers

The first step in sample preparation is isolating target proteins from their source, cells or tissues via lysis. Choosing the appropriate lysis buffer is the first step of your WB to success. The two major considerations are whether the chosen antibody will recognize denatured samples and the expression location of your protein of interest. Lysis buffers differ in their ability to solubilize proteins, with those containing sodium dodecyl sulfate (SDS) and other ionic detergents considered to be the harshest and therefore most likely to give the highest yield.

Cellular Location Buffer Recommended
Whole cell NP-40
Nuclear / Mitochondria RIPA, or nuclear /mitochondria fractionation for increased protein of interest concentration
Cytoplasmic (soluble) Tris-HCl buffer
Cytoplasmic (cytoskeletal bound) Tris-Triton buffer
Membrane-bound NP-40, RIPA or Tris-Triton buffer

Common Lysis Buffer Recipes

  1. NP-40 Buffer
    • 150 mM NaCl
    • 1.0% NP-40 or Triton X-100
    • 50 mM Tris pH 8.0
  2. RIPA Buffer
    • 150 mM NaCl
    • 1.0% NP-40 or Triton X-100
    • 0.5% sodium deoxycholate
    • 0.1% SDS
    • 50 mM Tris, pH 8.0
  3. Tris-HCl Buffer
    • 20 mM Tris-HCl, pH 7.5
  4. Tris-Triton Buffer
    • 10 mM Tris, pH 7.4
    • 100 mM NaCl
    • 1 mM EDTA
    • 1 mM EGTA
    • 1% Triton X-100
    • 10% glycerol
    • 0.1% SDS
    • 0.5% deoxycholate

Protease and Phosphatase Inhibitors

Immediately following cell lysis, proteolysis, dephosphorylation, and denaturation begin to occur. These events can be slowed down significantly if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer. Ready-to-use cocktails of inhibitors from various suppliers are available.

Inhibitor Specificity of Inhibitor Final Concentration in Lysis Buffer Stock (store at -20°C)
Aprotinin Trypsin, Chymotrypsin, Plasmin 2 µg/mL Dilute in water, 10 mg/mL. Do not re-use thawed aliquots.
Leupeptin Lysosomal 5-10 µg/mL Dilute in water. Do not re-use thawed aliquots.
Pepstatin A Aspartic Proteases 1 µg/mL Dilute in methanol, 1 mM.
PMSF Serine, Cysteine proteases 1 mM Dilute in ethanol. You can re-use the same aliquot.
EDTA Metalloproteases that require Mg2+ and Mn2+ 5 mM Dilute in dH2O, 0.5 M. Adjust pH to 8.0.
EGTA Metalloproteases that require Ca2+ 1 mM Dilute in dH2O, 0.5 M. Adjust pH to 8.0
Sodium Fluoride Serine/threonine phosphatases 5-10 mM Dilute in water. Do not re-use once defrosted.
Sodium orthovanadate Tyrosine phosphatases 1 mM Dilute in water. Do not re-use once defrosted

Preparation of Lysate from Cell Culture

Preparation of Lysate from Tissues

Determination of Protein Concentration

In order to ensure equal loading of each lane, determination of protein concentration is important. There are some commonly used protein assay methods to determine protein concentration in biochemical laboratories. According to your experiment conditions, you can perform one assay to determine protein concentration.

Sample Composition Protein Assay Not Recommended Protein Assay Recommended
Arginine-rich protein Bradford assay Lowry, BCA or UV assay
Cysteine-rich protein BCA assay Lowry, UV or Bradford assay
Tryptophan- or tyrosine-rich (no extinction coefficient) UV assay Lowry, UV or Bradford assay

Preparation of Samples for Loading into Gels

  1. Denatured, Reduced Samples
  2. To denature, use a loading buffer with the anionic detergent SDS, and boil the mixture at 100°C for 5 min. The standard loading buffer is called 2X Laemmli buffer. The 2X is to be mixed in 1:1 ratio with the sample.

    2X Laemmli Buffer Recipe

    • 4% SDS
    • 10% 2-mercaptoethanol
    • 20% glycerol
    • 0.004% bromophenol blue
    • 0.125 M Tris HCl

    Check the pH and bring it to pH 6.8

  3. Native and Non-Reduced Samples
  4. An antibody may recognize an epitope made up of non-contiguous amino acids. Under these circumstances, it is important to run a WB in non-denaturing conditions. Generally, a non-denaturing condition simply means leaving SDS out of the sample and migration buffers and not heating the samples. Besides, the reducing agents β-mercaptoethanol and DTT must be left out of the loading buffer and migration buffer.

Protein State Gel Condition Loading Buffer Migration Buffer
Reduced, denatured Reducing and denaturing With β-mercaptoethanol or DTT and SDS With SDS
Reduced, native Reducing and native With β-mercaptoethanol or DTT and SDS No SDS
Oxidized, denatured Non-reducing and denaturing No β-mercaptoethanol or DTT, with SDS With SDS
Oxidized, native Non-reducing and native No β-mercaptoethanol or DTT, with SDS No SDS

More WB Resource

Western Blot Protocols & Troubleshooting & Guide

Western Blot Protocol

Western Blot Troubleshooting

Protein Transfer from Gel to Membrane in Western Blot Assay

Running an SDS-PAGE Gel in Western Blot Assay

Western Blot Illustrated Assay

Stripping and Reprobing Protocol

Western Blot Storage Protocol

For research use only. Not intended for any clinical use.