I am pulling down p31 protein which is tagged by GFP. We are using double purification to get nicer result as possible. We use Nickle beads (because p31-GFP has 6-His tag) as 1st purification, then reprobe with GFP beads after elution with Imidazole.
I got higher band in IP comparing with IP-blot (40 kDa difference, so it's considerable). p31-GFP is ~55 kDa with normal blot. For IP-blot, I see nice band (~65 kDa) but it's ~105 kDa in the IP!
Any explanations?
Other problem, I can see very clear horizontal line crossing all the gel (in loaded and unloaded lanes!!) at 35 kDa. I suspected that's keratin, but I found in other resources that keratin doesn't look like that and it's rather like vertical stripes in one or all loaded lanes! So do you have any idea about that as well?
Hi,
for me keratin was ast 55kda and appears rahter as a strong horizontal smear. Do you use the same denaturation steps for input and IP samples? Are your proteins complexed and nor completely denatured?
Regards
Sven
Hi Sven
The strong horizontal lane is at 35 kDa!! and it' not smear, just clear and thick line crossing the gel!
Yes I am using the same denaturation steps for everything, I am using SDS-PAGE, so I should get rid of all protein complexes that bound with my target protein. I used harsh lysis buffer (500nM NaCl) as well. Also, after finishing the washing steps of the IP, I boiled the samples at 96C for 5 min. I think all of that should get rid of any protein complexes that bound with my target protein, right?
But how to check if my target protein is in complex after all these steps? by Mass Spec?
if yes, actually this is my main approach but I am trying to optimize my IP first to get nice Mass Spec results!
I am looking forward for your suggestions!
Best
Mohammad
hi,
mass spec would solve the question. But You should try oncce to do the gels with fresh buffers and thoroughly cleaned glassware. Thos should exclude the possibility of an unspecific band at 35kda
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