Hi,
Previously, I had done protein extraction from tissue (mice brian).
Now I need to extract proteins from cell lines (U87 MG). Can i follow the same protocol? If someone has a protocol or suggestions, kindly help.
Thanks
Hello Sumera,
The protein extraction protocol varies depending on the lab and researchers. Its really hard to suggest without knowing your previous protocol. However, for cell line the widely used protocol is using RIPA buffer with phosphostop and protease inhibitors or you can follow the buffer mentioned in below references. After application of buffer scrap the cells then triturate and spin to remove cell pellet. All conditions were mentioned in the following articles.
https://www.ncbi.nlm.nih.gov/pubmed/26806571
https://www.ncbi.nlm.nih.gov/pubmed/24882407
Thanks!
I am quite new to the cell culture thing. In fact i am learning it.
I am confused in using the volume of buffer for a specific cell count. Is there any rule?
Below is the protocol i followed for tissues:
Protein Extraction From Tissue:
The whole tissue lysates were prepared by suspension of 50 mg tissue in 100 μl of ice cold lysis buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 10 mM phenyl methyl sulfonyl fluoride (PMSF), 1 % d-Dithiothreitol (DTT)). The suspension was homogenized UP400S Ultrasonic Processor (Hielscher Ultrasound Technology, USA). The sample was incubated at room temperature for 1 hr followed by centrifugation (HERMLE, LabortechnikGmbR, Germany) at 14000 rpm at 4 ºC for 10 min. The supernatant was transferred to a new microcentifuge tube and stored at -20 ºC while the pellet was processed further by addition of 50 μl lysis buffer followed by repetition of treatment. Both supernatants were pooled and centrifuged at 14000 rpm for 90 min. The final supernatant was stored at -80 ºC until further use.
Hi
You have to standardise on your own laboratory experience.
Here is the protocol for isolation of protein from cells which we uses in our lab.
Protein Extraction from Cell Lines
Materials:
Prepare 1x Cell Extraction Buffer using the following formulation:
Cell extraction buffer formulation
· 100 mM Tris, pH 7.4
· 2 mM Na3VO4
· 100 mM NaCl
· 1% Triton X-100
· 1 mM EDTA
· 10% glycerol
· 1 mM EGTA
· 0.1% SDS
· 1 mM NaF
· 0.5% deoxycholate
· 20 mM Na4P2O7
· 1 mM PMSF
· Protease Inhibitor Cocktail
This Cell Extraction Buffer may be apportioned into 1x aliquots in micro centrifuge tubes and stored at –20°C until ready for use. Thaw on ice prior to extracting cells.
Processing Cells:
Estimate cell density: Suspension Cells: Enumerate suspension cells by counting in a hemacytometer. Adherent Cells: Estimate cell density by visual inspection under a microscope. Confluence levels of 70–80% are found to be optimal for many signal transduction studies.
Stimulate cells as desired.
Transfer the cells into clean 15 ml conical tubes: Suspension Cells: Aliquot the desired number of cells in medium into clean 15 ml conical tubes. Adherent Cells: Remove the cells from their vessel by scraping. Transfer the medium containing the detached cells into clean 15 ml conical tubes.
Collect the cells by centrifugation at 300 x g for 7 minutes.
Aspirate the medium.
Resuspend the pellet in ice-cold PBS.
Collect the cells by centrifugation at 300 x g for 7 minutes at 4°C.
Aspirate the PBS.
Lyses the cells by pipetting Complete Cell Extraction Buffer into each tube. We recommend using 1 ml of Complete Cell Extraction Buffer per 108 cells. It is important to note that this value may require optimization for each specific application.
Transfer the lysates to clean microcentrifuge tubes.
Vortex the mixture, then incubate the mixture on ice for 30 minutes, with occasional vortexing.
Clarify the lysates by centrifugation at 14,000 rpm (13,000 x g) at 4°C for 10 minutes.
Transfer the clarified cell extracts to clean microcentrifuge tubes.
The clarified cell extracts should be stored at –80°C until ready for analysis. Avoid repeated freeze-thaw cycles. In preparation for performing the assay, allow the samples to thaw on ice. Mix well prior to analysis.
Hope this helps you.
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