Cell Lysis and Protein Extraction for Western Blotting

All the steps for protein extraction from cells or tissue (fresh or frozen) must be performed at 2-8 °C. The following is the composition of one common lysis buffer that is used to prepare protein samples. Visit the Calculators page for a list of recipes for buffers and other Western blotting solutions.

RIPA buffer for protein extraction ready-to-use-solution (Product No. R0278)

• NaCl (S3014) 150mM
  
• Triton X-100 (T8787) 1%
  
• Sodium deoxycholate (D6750) 0.5%
  
• SDS (74255) 0.1%
  
• Tris-HCl (T1503) pH 8.0, 50 mM

Protease Inhibitors – Don't Lose Your Proteins During Sample Prep

Protein Extraction Protocol Steps

1. Discard the medium in culture dishes with cells and wash the cells using ice-cold PBS.
   
2. Discard the PBS, add ice-cold lysis buffer.
   
3. Scrape the cells using cold plastic cell scraper. Collect the cells in microcentrifuge tubes.
   
4. Agitate the contents in microcentrifuge tubes for 30 min at 4 °C.
   
5. Centrifuge the tubes at 16,000 x g for 20 min at 4 °C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.

Extraction of proteins from cells in suspension

1. Centrifuge the cell suspension at 2,000 x g for 5-7 min at 4 °C. The cells are collected at the bottom of the tube, discard the supernatant.
   
2. To the cell pellet, add ice-cold PBS and wash the cells by centrifuging at 2,000 x g for 5-7 min at 4 °C.
   
3. Add ice-cold lysis buffer to the cell pellet. Agitate the contents in microcentrifuge tubes for 30 min at 4 °C.
   
4. Centrifuge the tubes at 16,000 x g for 20 min at 4 °C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.

Extraction of proteins from tissues

1. Dissect the tissue of interest on ice. Transfer the tissue to round-bottomed microcentrifuge tubes and snap-freeze by immersing in liquid nitrogen.
   
2. For 5 mg tissue, add 300 µL of ice-cold lysis buffer and homogenize using electric homogenizer. Add additional 300-600 µL of lysis buffer during homogenization.
   
3. Agitate the contents for 2 h at 4 °C.
   
4. Centrifuge the tubes at 16,000 x g for 20 min at 4 °C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.

Normalize total protein concentration of samples

1. Take a small volume of lysate to perform protein estimation assay.
2. Determine the protein concentration of unknown samples by comparison with the standards, ensuring that the standard is diluted into the same buffer as the unknown samples. Protein estimation may be performed using Coomassie protein assay reagent (Product No. 27813), BCA assay, absorbance at 280 nm.
3. Transfer appropriate volume of lysates to microcentrifuge tubes so that all samples will contain the same total protein concentration.
   
4. Add adequate ice-cold lysis buffer to make up all the lysates to the same volume.

Mix samples with Laemmli loading buffer

The following is the composition of loading buffer required to prepare the samples for electrophoresis.

2X Laemmli loading buffer:

• Bromophenol blue (Product No. B5525) 0.004%
• 2-mercaptoethanol 10%
• Glycerol (Product No. G5516) 20%
• SDS (Product No. 74255) 4%
• Tris-HCl (Product No. T1503) 0.125 M

1. To a volume of cell lysate, add equal volume of loading buffer.
   
2. Boil the above mixture at 95 °C for 5 mins. Centrifuge at 16,000 x g for 5 mins.
   
3. These samples can be stored at -20 °C or may be used to proceed with gel electrophoresis.