I am using RAW cells in recent experiment now. However, in previous experiment, when I used 6-well plate for treatment (adding 80ul RIPA buffer into each well), the protein concentration was too low to be detected. I have checked my plate and nothing was left on the plate. At that time, I had no choice but loaded all of the protein I harvested to run the western blotting. THis time, in order to avoid similar situation, I used 10-cm dish to culture RAW264.7 cells. Before harvest, the cell confluency is around 70-80% only. Therefore, I added 120ul RIPA buffer to each 10-cm plate to harvest the cells. In previous experiment, 150ul RIPA buffer was suitable for a 10-cm RAW264.7 dish at 80-90% confluency. HOwever, I still could not get adequate protein concentration using Lowry methods. Since my colleagues in the lab have no similar issues, the reagents we used should be ok. RIPA buffer was freshly made each time before harvesting cells. THerefore, I am wondering whether it was because of overdilution of protein or other possible causes. HOw to estimate the amount of RIPA buffer needed for harvesting protein might be an important question to for this experiment.Does anyone know how to estimate this? Or some other possible reasons might be helpful to solve this problem. Thanks for everyone's help!
120µl for lysing a 10cm dish is a tiny volume and I'm not sure you could take less. When using RIPA buffer, I was used to take between 500 and 1000 µl per plate without any problem for western blot analysis. Once again, there's no strict rule but depending of your protein of interest, loading in the range 50-500µg of protein should be largely, except if your protein is really weakly expressed. But I would definitly go for a western blot issue rather than a problem with the volume of RIPA you used. I hope this will be helpful.
Good luck!
After I scraped the cells, I put it in 4 degree cold room for 10 minutes. Then, I used sonification at 4 degree Celcius for 8 minutes. (As mechanical lysis). I think the issue that I encountered recently might be related to lower cellularity at the time of treatment/ harvest. Because, after that time, when I started treatment at higher cellularity, I got good results. So, probably it was related to low cellularity. THanks for your recommendations. I will check them during subsequent experiments.
Hello everyone... Could you please tell me how much amount of RIPA should be used for cell suspension (PBMCs). It's written in protocols that 100ul RIPA buffer should be used for 1x 10⁶ cells. I have counted cells and my cell count is different for different samples like for one sample it's 0.9 x 10⁵ cell. So, how to calculate the amount of RIPA for different samples? Any suggestions.
@sufaya jameel you can use 100ul of lysis buffer till 1×10e6 to 0.8×10e6 cells, lower than this cell density till 10e5 cells you must use 50ul of lysis buffer to get good protein concentration.
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