I am working with SH-SY5Y cells and differentiating them into neurons using retinoic acid. I need to extract protein from them in order to conduct a western blot, however, I am having trouble getting enough protein. I am using 6 well plates and seeding 250,000 cells per well, with 3 wells per condition and harvest/differentiate at 80-90% confluency. Once they are differentiated I place the plate on ice, wash with 300ul of PBS per well and then add 300ul of RIPA buffer and protease inhibitor solution. I leave this on there for a few mins and shake the plate before using a cell scraper to lyse the cells (scraping about 30 times). I then transfer all 3 wells worth of cells to a 1.5ml eppendorf and centrifuge at 14,000g for 15mins, I do not set a temperature, but by the end of it, the centrifuge cools to 4 degrees celcius. I then transfer the supernatant to another eppendorf and perform a BCA assay. My issue is I am getting very low/undetectable levels of protein and don't know what I can do to increase this. If anyone has any suggestions that would be greatly appreciated, thankyou in advance!
Hello Olivia Haller
Below are some suggestions which you could incorporate in your protocol.
1. Once they are differentiated, I place the plate on ice, wash with 300ul of PBS per well.
Wash the cells twice with 300ul of ice-cold PBS per well. Take care that the cells do not detach from the substratum.
2. Then I add 300ul of RIPA buffer and protease inhibitor solution.
Then you may add 150-200ul of ice-cold RIPA buffer instead of 300ul and add protease inhibitor solution as usual. Reduce the volume of RIPA buffer accordingly if you need a higher protein concentration.
3. I leave this on there for a few mins and shake the plate.
Incubate the plate on ICE for 20 minutes with occasional shaking every 5 minutes. In an intact cell, protease and phosphatase enzymes are typically sequestered in cellular compartments. However, during the process of cell lysis, these compartments are broken open, releasing proteases and phosphatases that may degrade proteins and cause changes in phosphorylation states. Placing the cells on ice during lysis will help slow down these processes.
4. Before using a cell scraper to lyse the cells (scraping about 30 times).
RIPA lysis buffer is a cell lysis solution that is used for total cell lysis, and then you may make use of a cold plastic cell scraper to scrape the cells off the surface of the plate. You need not use the scraper 30 times to lyse the cells.
5. I then transfer all 3 wells worth of cells to a 1.5ml Eppendorf and centrifuge at 14,000g for 15mins.
Perfect. You may centrifuge in the range of 12000-14000g for 10-15 mins but at 4 degree C.
6. I do not set a temperature, but by the end of it, the centrifuge cools to 4 degrees C.
No, this is an incorrect practice. Set the temperature of the centrifuge at 4 degrees C in advance so that the temperature reaches 4 degree C when you need to carry out the process of centrifugation.
7. I then transfer the supernatant to another Eppendorf and perform a BCA assay.
Perfect.
Just make these changes and I hope you get better results.
Best.
After cell scraping, harvest the cells. Resuspend the pellet in RIPA or any mild lysate buffer (with appropriate protease inhibitors) and incubate on ice for 30 minutes with constant agitation. Alternatively, vortex the tube every 5 minutes.Centrifuge (~12000 rpm for 20 min at 4 degrees Celsius) and collect supernatant to complete the process
Hi Olivia, you're welcome. That's great news ! Wish you all the best!
Malcolm Nobre