Immunoprecipitation - Protein A and G doing strange things (Jan/09/2006 )
Hi,
I've just started doing IP and When using protein G sepharose I find I always get a huge band on a western at about 30 kD I always assume this was the light IgG chain however when I did a control of just beads and lysate no antibody the band is still there. I even went as far as to just take some beads and boil them in SDS loading dye and again the band is there. Any ideas what it could be? Is it possible for protein G to come off its beads? am I treating them too harshly when boiling? I also tried similar controls with protein A sepharose and saw the same thing but at a higher molecular weight so its not that the beads have something wrong
Thanks
Hannah
Hi,
this is strange.
I was boiling protein A sepharose in laemmli buffer for 2-3 minutes.
Then I was blotting with protein A-HRP (because Ig from IP were also transferred to the membrane, but as they were no more native they were not recognized by protein A)
I have no extra bands.
do you block your beads with something? with BSA which would not be Ig-free, or with FCS?
Hi Hannah,
I've seen this before mainly with Protein-A sepharose. The protein A or protein G can leech off the sepharose beeds and will run at about 35 and 30 kDa respectively. You have already tried what I was going to suggest and try the other, given that both are leeching/degraded could it be the way you store the sepharose beads or the age (will leech with age). I would try and borrow a small amount from another lab and see what happens. I once received a bad batch from a company and did all the controls sent them the gel and they refunded the purchase.
Good luck,
Scott
> I also tried similar controls with protein A sepharose and saw the same thing but at a higher
molecular weight so its not that the beads have something wrong
which molecular weight does it have?
i find in my IP's a band at about 47 kD...
molecular weight so its not that the beads have something wrong
which molecular weight does it have?
i find in my IP's a band at about 47 kD...
Its hard to tell exactly where the band for protein A is due to the type of markers I use but its between 25 and 37 kD so i'd say roughly 30-35. I've never seen anything around 47KD
I've seen this before mainly with Protein-A sepharose. The protein A or protein G can leech off the sepharose beeds and will run at about 35 and 30 kDa respectively. You have already tried what I was going to suggest and try the other, given that both are leeching/degraded could it be the way you store the sepharose beads or the age (will leech with age). I would try and borrow a small amount from another lab and see what happens. I once received a bad batch from a company and did all the controls sent them the gel and they refunded the purchase.
Good luck,
Scott
Thanks scott! its reassuring to know that someone else has seen this before and that I'm not going mad. I don't think its a storage thing because I keep the beads in the fridge where they say to keep them. It could be an age or batch thing but I did borrow some beads off someone else and still saw the same problem having said that I don't know how old their beads were.
Hi Hannah,
Just thinking about it, if I am right I suspect the Protein A/G is coupled using CNBr coupling. I'm not 100% sure but I don't think that is completely stable in the presence of ionic detergents such as SDS (prehaps a protein guru could help out here). Given that it is a common procedure I suspect it is not a problem when in normal SDS loading buffer but you may want to check out your loading buffer, it may have a higher concentration of SDS then you expect. My 1 xSDS gel-loading buffer (standard lamelli) is 50mM Tris-Cl (pH6.8), 100mM DTT (added fresh), 2% SDS, 0.1% bromophenol blue, 10% glycerol.
Or you could try someone elses loading buffer.
Just an idea, hope it helps.
Scott