help! non-specific background in Co-IP - (Aug/14/2009 )
I'm using protein A to determine the interaction between two proteins and always got non-specific bands in irrelavant IgG (rabbit IgG)control. i changed the lysate buffer to BSA (1mg/ml) lysate buffer and it helps but still not acceptable. anyone has better way to do it? please help me.
experiments conditions:
lysate buffer: 10mM Tris Hcl PH 8.0, 150mM Nacl, 1%NP40, 0.5mM Macl2, 0.15mM Cacl2 with 1 mg/ml BSA.
lysate cells in lysate buffer for 30 mins. preclear lysate with rabbit IgG, protein A together for 1h. split lysate into rabbit IgG for control and anti-flag (tag for my protein). incubate at 4c for 4 hours. wash three times with BSA lysate buffer. boil beads and run western.
How many bands and what size? Are they the same intensity as your desired bands?
How much lysate are you loading in your IP?
Have you tried blocking your beads?
miBunny on Aug 15 2009, 10:16 AM said:
How much lysate are you loading in your IP?
Have you tried blocking your beads?
Thanks for reply!
my desired band is around 33kda. and this non-specific band is just the same size as my desired band. funny thing is that i tried to transfect 293T cell just with the empty plasmid (pcDNA3.1) and run the Co-IP and there's a band around the same size even in the empty plasmid group. the non-specific band is not as bright as my desired band. but it's always there and can not be removed.
i used 500ul lysate buffer to lysate cells and after boiled the beads, i loaded 15-18 ul samples to the gel.
i haven't tried to block my beads. when i use BSA lysate buffer the situation seems to be improved but still got non-specific bands. i'm a little worried if i block the beads over night it would also interfere the interaction of the Ab binding to beads.
Do you see a band around 50 kDal? You may be picking up the heavy and light chain from your IP. The 33 kDal is around the size that light chain runs (around 25 kDal give or take a few). What species is your IP antibody and what species is your primary (western blotting) antibody?
If that is the case, you can use an HRP-conjugated protein A or protein G as your secondary for western. The protein a and g can't recognize linearized antibodies and will only bind to your primary antibody.
Do you see the nonspecific bands with your lysate control? If so, you may want to also work on your western conditions.
You can also try playing around with your blocking conditions to clean up your western. Try 3% ovalbumin alone or with the BSA.
Also, decreasing the amount of primary and doing an overnight incubation at 4 degrees can cut down background bands.
miBunny on Aug 16 2009, 02:36 AM said:
If that is the case, you can use an HRP-conjugated protein A or protein G as your secondary for western. The protein a and g can't recognize linearized antibodies and will only bind to your primary antibody.
Do you see the nonspecific bands with your lysate control? If so, you may want to also work on your western conditions.
You can also try playing around with your blocking conditions to clean up your western. Try 3% ovalbumin alone or with the BSA.
Also, decreasing the amount of primary and doing an overnight incubation at 4 degrees can cut down background bands.
my IP antibody is anti-rabbit. primary antibody is anti-mouse.
is HRP-conjugated protein A a totally different product than normal protein A? i need to buy it from company?
there's no non specific bands in my empty plasmid transfected cell lysate control.
by blocking conditions, do you mean the block step in western or the block step for beads before IP?
If your lysate is clean, you may be picking up the chains from your IP antibody. HRP-conjugated Protein A is normal Protein A that has horseradish peroxidase conjugated to it. Protein A only recognizes antibodies when they are in the native form (not denatured on a western blot) so it is a handy trick to cut down on light chain and heavy chain background.
I know Sigma carries it and probably some other companies.