Vertical streaks on WB following IP - (Jun/19/2008 )
I have recently experienced problems while trying to IP a GFP-tagged, multiple-pass TM protein from transiently transfected cells (~190kDa). I have tried this IP twice, each time getting messy vertical streaks down my IP lanes. I use the following protocol:
1. Transfect 60mm dish with GFPTagged TM protein
2. After 48 hrs, wash cells 2x with PBS before lysing
Lysis buffer: 50mM Tris, pH 7.4, 1% NP-40, 150mM NaCl, 5mM EDTA
3. Pass lysate (1ml) through 27G needle 8x, clarify lysate by centrifugation (10' at 12000rpm)
4. Add 1ul rb anti-GFP to lysate, rotate 2hrs at 4C
5. Add 25ul bead bed volume protein A beads, incubate 2hrs at 4C
6. Wash beads 3x with lysis buffer
7. Elute protein from beads (70C for 10' - from a paper where they IP'd the same protein). 150 ul elution volume.
8. Run 5%, 10%, 25% and 50% eluate on a 7% SDS-PAGE
9. WB using same antibody used for IP (I have no other choice)
When I develop my WB, I have messy vertical streaks in only the IP lanes and none in my input lanes. In the lanes loaded with less eluate (5%, 10%) I can make barely make out some bands, but most of it is lost in the streak. The streaks start at the top, where the resolving gel started, and continue the entire length. I believe there are bands within the smears at the 250kDa and 200kDa marker that correspond my protein. I have done many successful IPs (mostly for cytoplasmic and single TM proteins) without pre-clearing so I figured it should not be a problem... I have remade all my protease inhibitors in case degradation is a problem. I have also placed an order for protein-A HRP to eliminate the antibody species problem. Instead of heating my sample, I might try eluting with glycine pH2.5 to prevent aggregation of my protein. Is there anything else you can think of that might have caused the horrible streaking?
The messy smear is a result of using the same antibody to IP as to Western.
I have seen this myself. It is important to change antibodies.
The smearing can be reduced a little by doing a final, short wash in 1x TE
to remove the detergents.
But if you are IPing and Western blotting with the same antibody you will
always have huge problems unless you overexpress your protein to incredibly high levels.
Thanks for the reply! I think the smears are due to my GFP antibody. I have repeated the experiment, this time using my TM protein tagged with GFP and FLAG (on opposite termini) and IP'd with anti-FLAG-M2 beads. After transfer, I can see a very distinct band in my IP lane by Ponceau. When I probe the membrane with anti-GFP, I get the smears again. Perhaps the GFP antibody has gone bad or maybe it just does not perform well on WB following IP.
I regularly IP with the same antibody I blot with and can get good blots without streaking. A few times I had streaking like you describe and for us at least it seemed to be linked to accidently bringing a few agarose beads into the lane when loading.