I am providing my western blot image developed by ECL. Lane 1 is the marker and Lane 2 is my recombinant His-Tag Ni-NTA purified (control +ve, 24KDa). Lane 3 and others are the lysates loaded after proper lysis. The protein size is higher by about 3KDa in lysates loaded. Is it because some PTM is involved? Since not much data is available for the protein I am dealing with, how am I to interpret these results? Should I go for the MS of the protein?
Did you checked the sequence of gene cloned. Some times it was observed that some of the proteins have high level interaction with the other molecules in lysate like metabolite and proteins or peptides. So there is two possibility.
1. Interaction, if you cloned proper gene
2. PTM. Try to check the protein sequence, probably you can get idea about PTM, for example number of amino acids for phosphorylation and or any other modification. You can identified these modification by MS.
Hi,
@Sawan Jha, I expressed the protein in BL21(DE3)pLysS.
@Narendra Sharma, I confirmed sequence cloned into the vector and it is fine. I have used netphos and it predicts phosphorylation sites as: Ser: 15, Thr: 2, Tyr: 3. However, since there are no signal sequences in my protein, it is theoretically unlikely to undergo glycosylation.
1. what protein is it? 2. what are the lysates you are running, also from the same bacteria? Perhaps, the purified pos control has undergone some degradation event on the other terminus during the long purification procedure, whereas the lysates were made just prior to loading the gel? We need some more information here.
You need to know that your positive control is correct, first of all. We would take that 24kDa control protein and quickly check an accurate mass on ESI-MS. Only then can you worry about the biological samples. Also, your control is Histagged -we often find anomalous migration for these - it might be that your standard is wrong and your biology is right!
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