I’m attempting to co-IP binding partners from a protein. The problem is that no matter what I do, I have ”smudgy” lanes with super high background. This is a problem because I can’t see if my bands of interest appear. I am using near-infrared detection using a Li-cor odyssey scanner and the background appears in both channels, no matter which specific secondary I use.
Protocol info:
My IP protocol is pretty standard from what I can tell. I use pre-cleared GammaBind sepharose beads. Anti-myc antibody to pull down my protein of interest. Elute with a SDS sample buffer that contains bromophenol blue, DTT, B-ME, and various inhibitors. Boil at 95C for 7 minutes, spin down beads, and load the supernatant for SDS-PAGE. I transfer to a nitrocellulose membrane and do pretty standard Western procedures after that.
Troubleshooting so far:
- different beads (protein A/G Plus from Santa Cruz)
- different sample buffer containing different dye (Li-cod’s own with Orange G)
- loading beads that don’t have antibodies doesn’t help
- it’s not a heavy/light chain or antibody compatibility problem
However, NOT boiling the beads at all after adding sample buffer seems to reduce the effect a lot. Still, I really want to know what the problem stems from. We know of other labs that use near infrared detection with the same equipment and very similar protocols and have great success, and they boil their samples. Any suggestions or ideas would be greatly appreciated!
Do you a smear with your input as well? i.e. s your signal specific to your IP?
Have you tried probing with a no primary Ab western blot control? Alternatively, using Tubulin signal as an additional IP control (your IP should not contain TUBB signal unless of course your protein binds TUBB ) should help diagnose the problem.
Hi Sébastien Soubeyrand ,
There isn't any smearing in my input lanes, the bands look nice. The smearing is only in my IP lanes and negative (no pulldown antibody lanes).
I actually have tried using no primary Ab in the Western. One of my troubleshooting attempts involved just adding sample loading buffer to the beads, boiling, spinning off the beads, and loading that supernatant into the gel - no protein or antibody, and no primary on the Western, just incubating the membrane with secondary after transfer. In the other lanes I loaded the same thing but with no boiling, just antibody in another lane (in case the pulldown Ab was causing background somehow), and MW marker. I saw huge background in my bead lanes that were boiled, and very little in the no boil bead lanes.
That, coupled with the lack of background in my input lanes, makes me think something about the beads themselves when boiled causes the background, even though I don't actually load the beads. Maybe something is leeching off of them into my sample buffer prior to loading? I tried different beads and a different sample buffer so I'm lost.
HI Mohammad Amin Ghane
I am doing IP experiments these days and probably will be doing Co-IP as well. However, I have found it difficult to remove the background from the Rabbit IgG (Negative) lane. I IPed the protein of interest with an equal amount of antibodies, however, the lysates were in low concentration for the negative lane and I found a little decrease (promising) in the background.
Maybe you can also try to reduce the initial protein amount and go on without boiling the mix as you stated.
I would certainly like to try this thing next time I do my IP and/or Co-IP and let you know. Do you mind sharing any picture of the experimental result
PS: Great question and troubleshooting applied though!!
A pic would help. Do you see a smear that runs the entire lane? Does it stain with ponceau? If you see an intense signal, likely that your secondary reacts with an abundant epitope. Are you using ECL or fluorescence?
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