Hi there,
I am doing western blot for PPAR gamma in 3T3-L1 differentiated cells.
Since, its an adipogenic marker expected a good band in differentiated 3T3 cells.
every time I repeated the western blot I've got complete blank blot. I tried changing the concentration of antibody at the max to 1:300, It always showed empty blot. Besides, did some more adipogenic markers but did not get any bands. I stripped the same blot and added B-Actin to it and it shows good band. So would say there is a transfer of proteins to the blot.
(the PPAR antibody was brought from CST)
Some details about sample preparation:
Prepared samples with RIPA Buffer for the 1st time and then even changed the sample preparation with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents
Still don't get any bands.
Could anyone suggest me what can be the problem?
Thanks in advance
Farhath
Hi, PPAR gamma is a pretty big protein, in the same order than beta actin so your transfer should be good, However, you could use Ponceau R to immediately detect the proteins on your membranes, along with your size marker, that would confirm that you have proteins > than 50 kDa.
For the antibody, I would recommend that you use a positive control to test it. Also, has it been used before on these cells? Did you replicate a protocol you found in the literature? You can use milk or BSA, sometimes both don't work. Also, are you using the good secondary antibody? WB can be tedious if protocols are not in place as there are so many ways to perform it. Having these information would help greatly.
Best,
Hey Marie,
Thanks for your response. Usually I do Ponceau R staining though I don't see any bands I did get results. So don't rely much on Ponceau stain.
I am working with these cells for the 1st time and usually use milk. Just because I dint get results I have started using BSA too.
Yes,I am using the right primary and the secondary for no doubt.
Question: Is there any specific way to prepare the protein extract for 3T3-L1 cells?
Thanks again,
Farhath
I am not familiar with 3T3 L1 cells protocol, however, your answer about Ponceau is concerning. Did you not leave it long enough? Ponceau marks all proteins, at least your marker should be clearly visible. For me, that should be your first step to obtain results.