I lysed 1 million PBMC in 100µl and 200 µl of a commercial cell lysis buffer (cell signaling) and then I measured the concentration of proteins with a Bradford assay.
I compared two different volumes because I have no idea if it is going to be very diluted or not. I was surprised when I obtained the same concentration! I was expecting a difference in concentration between the 2 samples...
Is 1 million PBMC enough? or I need to reduce my cell lysis buffer volume?
Thank you!!!
Dear Yara,
Your question lacks some details. What is the use of the protein (WB, ELISA, etc.)? What was the apparatus that you used for protein quantification, since certain devices has a detection range and if your samples have concentrations out of the range, the device cannot measure their concentration accurately. By the way, 1 million cell per 100 ul seem quite proper for routine uses. Also, I recommend you that your sample: lysis buffer ratio not exceed 1:9. Thus, you avoid excess dilution or if needed (according to your experiments) you can easily add proper buffer to do further dilution.
Hello Yara
One million PBMC is not sufficient. I suggest you lyse around 6-7 million PBMC and reduce the cell lysis buffer volume. You can use half the volume of the lysis buffer (100ul) which will help you to obtain a good concentration of proteins. I hope the lysis buffer contains anti-proteases cocktail and phosphatase inhibitors.
Regards.
Hello, for our unique assay to measure kinase activity within a lysate we have compiled a protocol for sample prep of PBMC's. Please look at our resourche page and you might find details that are usefull. Check out, www.pamgene.com/ps12
Email me if you have any questions, [email protected]
1x10^6 PBMCs in 100ul of lysis buffer plus proteases/phosphatases inhibitors is sufficient for WB to detect a wide range of proteins, also phospho proteins. If you had the same concentration you might need to review the way you are doing it, I mean, pipetting and breaking the cells to get the proteins. What is the buffer you are using?
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