For some months we have been trying to optimize several immunoprecipitations in order to achieve higher recoveries. So far our immunoprecipitations are very specific for our proteins of interest but unfortunately yield very low recoveries.
For example, we have been immunoprecipitating albumin out of HepG2 cell lysates (500 μl lysate; +- 10 mg/L albumin) with mono- and polyclonal antibodies. When eluating this in different elution buffers (25 μl) we end up with the same concentration (10 mg/L) whilst concentrating almost 20 times, suggesting only recoveries of about 5 - 10%.
In order to perform experiments after IP (e.g. MALDI-TOF) we are seeking for concentrations of at least 100+ mg/L.We have been trying several protocols found in the literature, and additionally we tried to perform several optimalisations:
- Protein A versus G
- Direct vs indirect IP
- Incubation times
- Lysis buffers
- Antibody concentrations
Unfortunately, none of these changes would yield us significant higher recoveries.
Would anybody have an idea on how to approach this? Could anybody point me to critical points in order to achieve a high yield as possible? Preferably we are looking for antibody-based solutions.
If you simply want to detect the protein of interest, your best approach is to experiment further with your lysis buffers. Any polyclonal antibody (affinity purified against the antigen) is your best tool to start with. I recommend to lyse your cells in 1% SDS in RIPA buffer, and dilute the lysate 10 times (or dialyze to bring down to 0.1% SDS in RIPA buffer) before adding the antibody. In addition to the SDS, the RIPA should contain sodium deoxycholate and NP40. Albumin is a tough protein to work with, as it contains lots of other molecules that will co-precipitate, even under mild lysis conditions. These interactions prevent optimal binding to antibodies. This is why a harsh lysis approach is probably required, but make sure to not inactivate your antibodies by such harsh lysis conditions.
First I agree with the above suggestion… I don’t understand…are you setting up a protocol to IP your protein of interest using another target? How do you calculate your % of recovery? I mean is it by comparing to the total estimated in the input? If yes then you are losing it somewhere, probably in the flow through. I am sure you’ve tried very harsh condition to elute…
If you need more recovery then either scale up (use several IPs and pool them) or use tandem IP (i.e re-IP the flow through) until you don’t detect any more of your protein in the FT (~ immunodepletion). If necessary concentrate the flow through using Amicon with appropriate membrane pore cutoff…or if you detect IgGs in the FT then use more protein A/G. If you can estimate the concentration in moles of your protein in your input you may have an idea of how much antibodies to start with… Increasing the amount of antibodies in the IP may or may not help. Excess antibodies can result in binding competition which will lower the recovery...I
Concentrating your protein is not a big deal. You can try lyophilization, etc...There are a plenty of methods you can use depending on your downstream application.
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