Good morning, I am a PhD student trying to get my head around brown adipose tissue western blot. So far I have successfully isolated proteins with high molecular weight such as PGC1a but I keep having poor results with ucp1. precisely, I can visualise my band but this is always accompanied by a smear that makes it harder to quantify. Also, my stripping buffer is unable to remove the antibody for me to do my beta-actin. Being at a very close molecular weight (especially considering the aforementioned smear) I did not manage so far to precut the membrane to sort out this problem.
I had the same issue with both the antibodies:
https://www.abcam.com/ucp1-antibody-ab10983.html
https://www.abcam.com/ucp1-antibody-epr23004-34-ab234430.html
Does anyone have any suggestion on what may have gone wrong? I've tried to control the buffer pH as well as doing multiple centrifuges to eliminate all the fatty residues, but the signal keep being extremely strong even at the lowest exposure time.
p.s I am using rat tissue.
Dear Arianna Fozzato, stripping the membrane and incubating it with another antibody solution might be not the best idea, since you are stripping ceratin amount of your protein of interest off the membrane as well. In addition, you might want to use total protein staining for your normalization strategy instead of housekeeping protein (beta-actin). Housekeeping proteins are often influenced by your experimental treatment or are up-regulated in specific cell or tissue types.
You might find this helpful: https://www.dyeagnostics.com/site/en/products/spl/
Dear Arianna Fozzato
I found from your image that the smear does not appear in some samples where you could find a clear band. Is there andy biological or technical difference between the samples that have or have not the smear? Accordint to sample preparation, I suggest to lyse samples in a simple RIPA buffer withoud SDS using a potter and pestel in ice. Use a proportion of 1 ml of buffer/100 mg of tissue so that the lysate will not be too concentrate and you can quantify by Bradford or Lawry. Denature the sample with 2X Laemmli buffer with 2-mercaptoethatl at 96 degrees for not more than 5 minutes or protein aggregates will form and will precipitate. Alway use fresh acrylamide and buffers to prepare the gels (open no more that 1 month earlier). Same for Running. Check the current settings when you start the run. If you are using an amersham system to run the mini-gel, I set the current at 20 mA/gel and the voltage shoud start between 60 and 80 if all it is ok. Finally, do you see the smear also in the ponceau staining? or it is only the ucp1 band? did you try with another protein at the same MW as GAPDH?
All the best.
Try to increase your centrifugation to 20 minutes at highest speed at 4C. Incomplete homogenization can leave particulates as well as filamentous DNA in the sample. Both of these hold up proteins from entering the stack evenly, leading to streaking. Also once your sure that you have removed particulates and DNA, do a titration. Overload can cause the matrix to be saturated and proteins can 'bypass' the matrix and run along the glass plate outside the gel. Overload is the most common cause of streaking. It can also effect the migration of proteins when resolved so they don't behave according to the reported MW.
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