I am trying to run an SDS-PAGE after his-tag purification of a sample. The protein of interest is an oligomerising membrane protein with an YFP tag attached. When the fractions of the sample from the different purification steps are analysed for YFP fluorescence, the samples show the expected profile (i.e.: high in cell lysate and final elution, low in washes, etc) and at high recovery rate of the protein.
However, when I try to run the samples in an SDS-PAGE, I do not get an extra band for the protein of interest, compared to the WT sample. There are no extra bands, so the cleavage of YFP from the rest of the protein is unlikely.
I am using 5% ß-mercapthoethanol in my SDS-loading buffer, and let the sample stand in the buffer for 30 minutes at 37C (no boiling). What could be the reason for this missing band in the SDS-PAGE?
Thanks for all help in advance.
Hi Marco Pedretti,
Thanks for your answer, and sorry for the confusion.
After the purification, the YFP-tag is NOT cut off. I only mentioned cleavage in my post, because that would explain an incorrect band showing up in the SDS-PAGE (for the YFP separately and the rest of the protein) - but this does not happen.
The molecular weight of the protein is 40kDa.
I previously boiled my samples (with identical results - no band), but was recommended not to do that as this is a membrane protein and aggregation can be an issue.
Hi Andras Sandor,
As what I understand from your question is that you have purified a his-tagged protein which also carries a YFP tag.
As you aren't cleaving the YFP tag, when you run this protein in SDS-PAGE, you will get a single band of your protein-YFP conjugate. So, which band is missing that you expect to get?
Lastly you are absolutely correct that certain membrane proteins aggregate upon boiling prior to SDS-PAGE. In that case try heating it to 70c for 15mins in sample loading buffer.
Hi Tushar Chakraborty ,
Thanks for your answer - you understand it correctly, there should only be a single band (which is the protein-YFP conjugate). That band is missing. However, I would expect it to be there, since the same samples fluoresce quite strongly when measured in a plate reader.
Thanks for the idea on changing the heating temperature - I will try that.
How are you detecting your protein? Using and anti-his antibody? Or detecting the YFP tag? Or another method? I would like to help you but I need this information because otherwise I may give conflicting / unhelpful statements.
Hi Sebastian Lühn ,
I am using InstantBlue to stain the proteins in the SDS-PAGE gel.
Ok, so anything reagaring tag accessibility for antibodies can be excluded as the cause for this. Since you are purifiying a membrane protein. Are you working with detergents or nanodiscs to stabilize it? Maybe a detergent causes some problems.
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