I recently extracted my protein from day0, day2, day4 and day6 cell culture and performed a BCA. I intend to carry out a western blot, however, the protein concentration obtained from the BCA of all the above days is too low.
What are some of the possible factors that could've influenced this to avoid the next time? Is there anything that I can do to increase the protein concentration?
Hi Magdaleena,
Perhaps you could lyophilize your extract and then resuspend it in a smaller volume in order to rise protein concentration and try to detect them by western blot.
Is that a validated expression method?
What is your extraction protocol?
Is there any positive control sample as a reference material?
If so, analysis of this sample can make it possible to identify minimum required concentration for WB and for comparison of your cell line & extraction recovery.
Thank you for the response. I will consider to lyophilize the protein, thank you María Luisa Caballero . İsmail Emir Akyildiz yes, this is a validated expression method. I didn't consider referencing a positive control sample but I can look into that..thanx! Harald Kühnel
, I used RIPA. I'm also considering that the quantity I used is what has influenced the results, but I am not sure. I will check into it further. Thank you, this was of great help
Hi Magdaleena,
There are several factors that could influence the BCA readings. I have listed a few here, and also included relevant questions for you: 1) Total number of cells you lysed, 2) Volume of buffer used for lysis, 3) Dilution of lysate in BCA assay, 4) Did you try increasing BSA concentrations (for standard curve generation) in the BCA assay?, 5) How old are your reagents?, 6) Do you want to try some secondary/alternative method to make sure you have a decent protein concentration to start with? One example may be found in the attachment (please also see references therein). This method will provide you quick results, but is only qualitative; BCA should be final/best approach for quantitation purposes.
Hope this helps.
Anoop
Anoop Arunagiri Thank you for your response. And thank you for the relevant questions to consider. I will take a further look into the attached folder you provided. This is all of great help.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts