Western blot of the supernatant - (Jul/17/2008 )
Hello,
I'm conducting a Fc-tagged protein purification which is secreted into the supernatant by a signal sequence.
I checked the supernatant (from normal 10% FCS medium), before performing the purification, with WB using Anti-mouse HRP (the only Ab used for developing the blot at 1:1000), and there was no protein in the SN. Although it might be that the PhCa transfection did not work this time, I am still getting a very fade "flattened" band at about 58 kDa in every lane.
Any idea what this might be?!
Attached is an image of 3 lanes, loaded with 3 different 10X-concentrated 45um-filtered supernatant, expected to contain 3 different Fc-tagged proteins (one in each lane), at approx. 37, 66 and 87 kDa (left to right). Exposure time is 96 minutes. 10% SDS-PAGE ran at 60V for 30 minutes, then at 120V for 75 minutes.
Thank you very much.
Hello again,
A colleague of mine told me that this might be a BSA band. I am not sure if this is correct though!
But I'm wondering, how it comes that this post hits 53 views without any comment.....?!!! 
Thanks for you all 
.
Can you check your transfection efficiency by cotransfecting with GFP or something?
Did you check your transfer efficiency?
Can you load a gradient of sample to see if its its overloaded?
Is it a polyclonal antibody?
Did you check your transfer efficiency?
I will, since this is a new batch of self-made HeBS buffer. My lab is running short of money, so we have to save on the commercial kit.
I loaded the unconcentrated SN, and 10X-concentrated SN.
What do you mean by "overloaded"?
Yes, it's a polyclonal Ab.
Thank you very much "Judes" for passing and replying to my post
. Hi newbio,
I am trying to do the same as you i.e. isolate an Fc-fusion protein from supernatant. I did a control tranfection with a GFP vector (not Fc-tagged). With Ponceau staining of my blot, I saw a band at around 60 kDa in the control as well as in the lane with the Fc-tagged protein. In the latter lane i saw other bands as well, which gave me hope as my protein is multimeric. I used a goat anti-rabbit HRP as my secondary Ab. It has bound nonspecifically to every protein band in the blot, including the ladder!
Anyway, I just replied to tell you that i too get this ~60kDa band that you are seeing.
Did you try to concentrate the protein in your superntant? I concentrated the proteins using centricon and microcon filter devices, before loading onto the Western blot.
Hello suds,
Thanks for sharing your case in here.
Actually, my colleague was right in her point about the BSA band, since one can get rid of it by using a serum-free medium. (but it is expensive though!)
And thanks for the tip; Yeah I concentrated my SN 10X; This time I ended up with an ugly gel, due to the high amount of salts (I think).
Greetings!