Sample Preparation in Western Blot Assay

Contents

• Lysis Buffers
• Protease and Phosphatase Inhibitors
• Preparation of Lysate from Cell Culture
• Preparation of Lysate from Tissues
• Determination of Protein Concentration
• Preparation of Samples for Loading into Gels

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.

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.

Preparation of Lysate from Cell Culture

• Place the cell culture dish on ice. Wash the cells with ice-cold PBS twice.
• Aspirate the PBS. Add appropriate ice-cold lysis buffer.

Note: 1 mL/ 10⁷ cells/100 mm dish/150 cm² flask; 0.5 mL/ 5x10⁶ cells/60 mm dish/75 cm² flask.

• Scrape adherent cells off the dish using a cold plastic cell scraper. Gently transfer the cell suspension into a pre-cooled microcentrifuge tube. Maintain constant agitation for 30 min at 4°C. Then, centrifuge in a microcentrifuge at 4°C.

Note: The centrifugation force and time depending on the cell type. A guideline is 20 min at 12,000 rpm.

• Gently remove the tubes from the centrifuge and place on ice. Aspirate the supernatant and place in a fresh tube kept on ice. Discard the pellet.

Preparation of Lysate from Tissues

• Dissect the tissue on ice as quickly as possible.
• Place the tissue in Eppendorf tubes and immerse in liquid nitrogen to snap freeze. Keep on ice for immediate homogenization.
• For a ~5 mg piece of tissue, add 300 μL of ice cold lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2 x 300 μL lysis buffer, then maintain constant agitation for 2 h at 4°C.

Note: Volumes of lysis buffer must be determined in relation to the amount of tissue present.

• Centrifuge for 20 min at 12,000 rpm at 4°C. Aspirate the supernatant and place in a fresh tube kept on ice. Discard the pellet.

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.

Preparation of Samples for Loading into Gels

1. Denatured, Reduced Samples

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

2. Native and Non-Reduced Samples

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.

More WB Resource

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Protein Transfer from Gel to Membrane in Western Blot Assay

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Western Blot Storage Protocol