All steps to extract protein from cells or tissues (fresh or frozen) must be performed at 2-8°C. Here are the ingredients of a common lysis buffer used to prepare protein samples. For more content, please visit Merck Life Sciences official website: www.sigmaaldrich.cn
ready-to-use solution for protein extraction (Classification No.: R0278)
NaCl (Classification No.: S3014) 150mM
Triton X-100 (Classification No.: T8787) 1%
sodium deoxycholate (Classification No.: D6750) 0.5%
SDS (Cat. No.: 74255) 0.1%
Tris-HCl (Cat. No.: T1503) pH 8.0, 50 mM
Protein Extraction Protocol Steps
- Cat.
discard PBS and add ice-cold lysis buffer.
Use a cold plastic cell scraper to scratch the cells. Collect cells in microcentrifuge tubes.
centrifuge tube at 4°C for 20 minutes. Collect the supernatant in a new tube and place it on ice. Discard the sediment.
Protein extracted from cell suspension
Cell suspension was centrifuged at 2,000G at 4°C for 5-7 minutes. The cells will gather at the bottom of the test tube and discard the supernatant.
To the cell pellet, ice-cold PBS was added and the cells were washed with centrifuged at 2,000 G for 5-7 minutes at 4°C.
Centrifuge the centrifuge tube at 16,000G for 20 minutes at 4°C. Collect the supernatant in a new tube and place it on ice. Discard the sediment.
Extract protein from tissues
Cut the tissue to be studied on ice. Transfer tissue to a round bottom centrifuge tube and immerse liquid nitrogen to quickly freeze.
For 5 mg of tissue, add 300 μL of ice-cold lysis buffer and homogenize using an electric homogenizer. Additional 300-600 μL of lysis buffer was added during homogenization.
Centrifuge the centrifuge tube at 16,000G for 20 minutes at 4°C. Collect the supernatant in a new tube and place it on ice. Discard the sediment.
Standardized sample total protein concentration
Take a small amount of lysates for protein estimation and determination.
protein estimation can be performed using Coomassie Protein Assay Reagent (Cat. No.: 27813), BCA assay, 280nm absorbance, or DirectDetect® infrared spectrometer for total protein quantification.
Transfer the appropriate volume of lysates to a microcentrifuge tube so that all samples contain the same total protein concentration.
Add enough ice-cold lysis buffer to make all lysates have the same volume.
Mix the sample with Laemmli loading buffer
The following are the components of the loading buffer required to prepare the electrophoretic sample.
2X Laemmli loading buffer
Bromophenol blue (Cat. No.: B5525) 0.004% 0.004%
2-mercaptoethanol 10%
glycerol (Cat. No.: G5516) 20%
SDS (Cat. No.: 74255) 4%
Tris-HCl (Cat. No.: T1503) 0.125 M
Add an equal amount of loading buffer to a certain amount of cell lysates.
The above mixture was boiled at 95°C for 5 minutes. Centrifuge at 16,000G for 5 minutes.
