Sample Preparation for Western Blotting: Cell Lysis and Protein Extraction

All the steps for protein extraction from cells or tissue (fresh or frozen) must be carried out at 2-8 °C. The following is the composition of one common lysis buffer that is used to prepare protein samples.

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

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

Extraction of proteins from adherent cells

  • Discard the medium in culture dishes with cells and wash the cells using ice-cold PBS.

  • Discard the PBS, add ice-cold lysis buffer.

  • Scrape the cells using cold plastic cell scraper. Collect the cells in microfuge tubes.

  • Agitate the contents in microfuge tubes for 30 min at 4°C.

  • Centrifuge the tubes at 16000G 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

  • Centrifuge the cell suspension at 2000G for 5-7 min at 4°C. The cells are collected at the bottom of the tube, discard the supernatant.

  • To the cell pellet, add ice-cold PBS and wash the cells by centrifuging at 2000G for 5-7 min at 4°C.

  • Add ice-cold lysis buffer to the cell pellet. Agitate the contents in microfuge tubes for 30 min at 4°C.

  • Centrifuge the tubes at 16000G for 20 min at 4°C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.

Extraction of proteins from tissues

  • Dissect the tissue of interest on ice. Transfer the tissue to round-bottomed microfuge tubes and snap-freeze by immersing in liquid nitrogen.

  • 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.

  • Agitate the contents for 2 h at 4°C.

  • Centrifuge the tubes at 16000G for 20 min at 4°C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.

Normalize total protein concentration across samples

  • Take a small volume of lysate to perform protein estimation assay.

  • Protein estimation may be performed using Coomassie protein assay reagent (Product No. 27813), BCA assay, absorbance at 280 nm, or using the Direct Detect® Infrared Spectrometer for total protein quantitation.

  • 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.

  • Transfer appropriate volume of lysates to microfuge tubes so that all samples will contain the same total protein concentration.

  • 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. L3771) 4%
Tris-HCl
(Product No. T1503) 0.125 M

  • To a volume of cell lysate, add equal volume of loading buffer.

  • Boil the above mixture at 95 °C for 5 mins. Centrifuge at 16000G for 5 mins.

  • These samples can be stored at -20 °C or may be used to proceed with gel electrophoresis.