Co-IP experiment a lot of problems!!!

Model Organism: Worms Methods & Reagents
1

I am trying to do Co-IP with the whole worm lyste for two proteins but I am facing a lot of problems.

  1. when I used preimmune serum as a negative control but instead of getting just the heavy chain band, I got a ladder like profile. Has anyone used
    Pre-immune serum for co-IPs.
  2. what should be the concentration of protein lysate to be used for Co-IP.
  3. and the amount of antibody to be used for IP.
  4. and the protocol for protein extraction as I am using sonication for the worm lysis (80% amplitude, 1 sec X6 times , 2 min. rest) and
    check under microscope till 90% worms are lysed.
  5. any other precaution to take.

kindly help.

2

Some more details would be useful:

Does your antibody work well enough just for a western on worm lysate?
Do you have a mutant or RNAi strain control w/ reduced or no protein present?
Do you have a transgenic over-expression strain?

What is in your lysis and immunoprecipitation buffer? Salt? Buffer? pH? Protease inhibitors? Divalent cations?
What is the protein concentration and ab concentration you are using?
Are you doing a pre-sorption step to non-immune or pre-immune antibodies followed by immune?
How many times do you wash the beads?
How do you elute?
Are you check in the flow-through after IP to see if your protein has been depleted?

3

Hi

  1. Yes my ab works very fine with just the western on worm lysate dilution used is 1:10,000.
  2. I do have a mutant strain with a truncated protein present but not detected by my Ab.
  3. No I do not have an over expression strain at the moment.
  4. I use HEPES lysis buffer for performing Co-IP ( working conc. 50 mM HEPES, 1mM EDTA, 10% glycerol, 150mM NaCl, and P.I cocktail pH-8.0)
    [but for westerns I use Tris 100mM, SDS 4%, DTT- 200mM, gLYCEROL 20%, bromophenol blue and boil worms at 100 degress for 10 min.
    and use the supernatant.]
  5. I used 3mg of protein for Co-IP, 2.5-3.0 ug of antibody for IP
  6. yes I perform the pre clearing step by nutating the protein A/G beads with the protein O/N at 4 degrees and then use the sup. for IP.
  7. I wash the beads 4 times 5 min. each (nutate at 4 degrees).
  8. I put 2x Lammeile dye boil the beads, spin down and load the sup.
  9. I also checked the F.T but the F.T was also Blank.
4

So Flow Thru was blank meaning you could see the protein in the extract to start, and all the protein ended up on the beads (disappeared from the lysate during IP)? Is the IP step overnight as well? I would presorb for ~1 hour, take off the extract and then do the IP another hour or so or overnight.

The ladder you speak of is a western blot w/ your purified antibody or is it a silver stain / Coomassie Blue stained gel or Ponceau S stained membrane after transfer?

-Kevin.

5

yes , there was protein in the extract which was confirmed by the BCA estimation as well as the normal coomassie gel that I ran,
and I guess what you suggest is right as there was no detection of protein in the F.T.

No the IP step is not overnight, The pre-cleared lysate was incubated
with antibody (3ug) for 1.5 hr at 4°C. 1/10 volume of protein A/G slurry was then added and incubated for 1.5 hr at 4°C. The protein A/G beads
were then washed with lysis buffer for four times and the bound proteins were eluted by boiling in 2X sample buffer for 5 min.

It was the western that gave me this ladder like profile (pre cleared lysate + pre-immune serum instead of the single 50 KDa heavy chain band
that should have been there.

Do you pre clear the serum before using?

Thanks

6

I’m a big fan of coupling my antibodies to beads using a cross-linker. Pierce sells a X-linker kit, although you can also just by the cross linker and follow the same protocol. You elute the same way, although I recommend omitting the reducing agent during the boiling in sample buffer. Otherwise, the IgG light-chain comes off which can be a mess around 25kDa. I still reduce, but after I pull the elute off the beads. I also use freshly added reducing agent, usually 2-mercaptoethanol (50 ul per 1 ml of 5x SDS-PAGE sample buffer). Fresh DTT is way more potent, but it’s also not as stable.

I guess I don’t have a sense whether the ladder you are seeing is from the worm extract or cross-reactivity with the IgG (in this case, probably your secondary ab).

7

Thank you Kevin
for your suggestions…
One more thing… have you used pre-immune serum for Co-IP and could you help me with the protocol you follow.
i.e if there is a step that I might be missing and what all precautions do you take while performing the experiment, so that I can get an idea.

8

Most of the IP’s I have done used affinity-purified antibodies from immune sera. There wouldn’t be much point to using pre-immune in that case, although you could use an equivalent amount of non- or pre-immune IgG (purified by Protein A/G agarose, for example).

I would just do the mock-IP in parallel, rather than doing the pre sorption step (although that works, too). So you’d make one extract (or two w/ one being the mutant; ideally made at the same time and never frozen), having the same concentration. Pull off the total samples from each for the western blot, then add the IgG coupled to the beads (control or affinity-purified), nutate for ~2 hours to overnight at 4°C, pull off the flow-through (take a sample of that), then wash the beads a few times like you did, and then elute in non-reducing sample buffer. Decant the elute, reduce with DTT or B-ME, and reboil. Then run the gel with all the samples, total (both lysates), flow through (both lysates, both IP’s), and IP’s (both lysates, both IPs). It’s a lot, but all the information will be there in that one experiment.

As for making the affinity-purified antibodies, that’s kind of up to you. I like injecting a His-tagged protein into the bunny, then making an affinity column with some other tagged version, like GST- or MBP-. This way you’re not just isolating anti-GST antibodies but only the shared thing that was in the two protein preps. I then couple the protein to a resin. Affigel 10 or 15 works well from BioRad if your protein doesn’t have cysteines, but if so, like a GST-fusion, you can use SulfoLink from Pierce. Couple according to the instructions, quench, and mock elute, typically 100 mM glycine, pH 2.5, and wash back into PBS, add azide and store until you’re ready for use. I usually add ~10 mg’s a protein to 1 ml of beads to make the column, store in PBS with some sodium azide, and you can reuse the column for years. Add your antisera (I usually add 1/10 volume of 10x PBS) + NaAzide, and mix for ~1 hour or so, wash with PBS, and then elute with 100 mM glycine, pH 2.5, as above. I take 0.5 ml fractions into tubes w/ 50ul of 1 M TrisCl pH 8.5 or so, to normalize the acidity. I usually take 5-10 fractions, find the peak by Bradford assay, and pool the peak. I usually cross link 100ug of antibody per ml of protein A beads according to the instructions in the Pierece kit, and I use 20ul of beads per IP. But your mileage will vary.

9

Thank you Kevin…
I will try to follow the protocol that you mentioned.

and Thanks for the insight too, that is definitely going to help me a lot!!

Best Wishes