Hello. I am a second-year master’s student and I am trying to validate a previously reported interaction between a bait and two target proteins through co-immunoprecipitation, looking for advice and troubleshooting options please. I have linked the figure of my final result (https://drive.google.com/file/d/1jve5Np582SLbkzn7hdFaz6H9uM9uRgLB/view?usp=sharing) where I have these specific problems:
1. Low bait and target expression in the input lanes. My input protein concentration was 3 µg/µL and I loaded 30 µg protein (20 µL) into each input lane.
2. Bands appearing at unexpected MW. In my bait and both target samples (I've only included one in the figure, but both look similar) I see strong signal around the 70 kDa marker. I previously generated positive controls for my proteins of interest, which are just high-yield transfected protein from Cos-7 cells, to confirm the expected protein MW. As indicated by the positive controls, my bait protein is expected between 70 and 100 kDa while my targets are expected just above 100 kDa.
3. Bands appearing in the empty vehicle IP lane. The strong B-actin band, included as a positive control for interacting with the bait protein, should not be present as there should be no pulldown in the empty vehicle-treated condition.
I will share more details on my protocol below:
On day 1 I split HaCaT cells (DMEM, 10% FBS, 1% PenStrep) from a confluent 10cm dish equally into two 15cm dishes. I allow the cells to reach 60-70% confluence over 24-48h, then change media and transfect them using X-treme GENE at a 2:1 transfection reagent:plasmid ratio. In my experimental dish I transfect 2 µg of my bait protein (not expressed in HaCaT, HA-FLAG-tagged) and 2 µg of each of my two target proteins (lowly expressed in HaCaT, sv5-/GFP-tagged respectively). In my control dish I transfect 6 µg of empty vehicle. To transfect I first add plasmid into my eppies, then 1 mL serum-free media, then transfection reagent and incubate at RT for 15m before adding directly into the media and swirling the plate. After 48h transfection I treat my cells with 10 µM MG132 to boost protein concentration then lift my cells with T/E and resuspend the pellet in 600 µL cold E1A lysis buffer (0.1% Triton X-100, 50 mM HEPES pH 7.0, 250 mM NaCl in dH2O; Triton added fresh before use) including 1x protease inhibitor. I then roll the samples at 4°C for approx. 20m then aliquot 10 µL for BCA protein quantification, 30 µL for the input, and the rest 560 µL for the IP. I washed the FLAG-tagged pulldown beads (70 µL) 3x in cold E1A + protease inhibitor, resuspended them with 70 µL cold E1A + protease inhibitor, then added 60 µL of the beads to each of my empty vehicle control and experimental IP samples. Then I rolled the samples (including input) overnight (16-18h) at 4°C. The next morning I discarded the supernatant and washed the beads 3x with cold E1A buffer, then boiled the beads in 60 µL boiling blue (Laemmli buffer, 15 µL for 3x gels) for 3 minutes and boiled the inputs in 30 µL boiling blue for 10m. I then western blotted for my proteins of interest using protein-specific primary antibodies and HRP-conjugated protein A/G secondary antibody.
I would appreciate any pointers on mistakes that I am making or how I can achieve a consistent result. Apologies for the long post, thank you for your help :)
** previous iterations of this experiment that I have also tried but yielded similar results include:
1. Transfect only bait protein (4 µg).
2. Transfect for 24h.
3. No MG132 treatment.
4. Boil IP samples for 10m.
Issues with immunoprecipitation (IP) experiments like absence of bands, wrong molecular weights, or non-specific bands in control lanes often stem from a few common problems related to antibody specificity, lysis conditions, or washing stringency. Here’s a systematic guide to troubleshoot and improve your IP:
1.
No Bands in IP
This can happen due to inefficient pulldown or detection issues.
Check these:
- Antibody quality: Use a validated, IP-grade antibody. Try a different lot or vendor.
- Antibody amount: Too little can yield no pulldown; too much can cause background.
- Protein expression: Verify expression by running a whole cell lysate (WCL) Western blot.
- Lysis buffer: Use a buffer compatible with your protein of interest (RIPA can be too harsh for some interactions).
- Bead binding: Make sure beads are compatible with your antibody (e.g., Protein A vs. G).
- Epitope masking: Avoid harsh lysis conditions that might disrupt protein structure.
- Input control: Always run an input (pre-IP lysate) to confirm protein presence.
2.
Bands at the Wrong Molecular Weight
This usually indicates:
- Non-specific binding: Your antibody or beads might be pulling down unrelated proteins.
- Degradation: Use fresh lysates and protease inhibitors.
- Post-translational modifications or isoforms: Your protein may run differently than expected. Run a positive control to verify this.
Tips:
- Include a molecular weight marker and a positive control sample.
- Check if your antibody has cross-reactivity by reviewing datasheets or using knockdown/knockout controls.
3.
Bands in the Empty Vehicle Pulldown (Negative Control)
This points to non-specific binding to the beads or the antibody.
Improve specificity by:
- Pre-clearing lysates: Incubate lysates with beads alone before adding your antibody.
- Blocking: Block beads with BSA or non-fat milk.
- Wash stringency: Increase salt concentration (e.g., up to 500 mM NaCl) or use a mild detergent (e.g., 0.1% NP-40) in your wash buffers.
- Include IgG control: Use a non-specific IgG as a negative control to assess antibody specificity.
4.
General Best Practices
- Optimize antibody:bead ratio: Start with ~1–5 µg antibody per 20–40 µL beads.
- Time and temperature: Incubate antibody with lysate for 2–4 h at 4°C or overnight for weak interactions.
- Elution method: Use appropriate elution buffer (e.g., Laemmli sample buffer with heating) and minimize harsh conditions unless necessary.
- Avoid overloading gel: Too much protein can cause smearing or non-specific bands.
Also, did you sequence the plasmids to be 100% sure they are the correct ones?
Never trust, always verify. It's so easy to grab the wrong tube from a freezer or get samples mixed it.
Michael Goldschagg Is there a problem with your protein sample? Is there a problem with the binding between the antibody and the protein? How is the transfer method? There are many factors that affect this, and it is a bit difficult to say clearly. You can first check whether there is a problem with the operation. If there is no problem, then check whether the antibody is abnormal.
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