Hello all,
I am encountering a weird problem in my immunoprecipitation experiments with GFP magnetic beads. I have co-transfected HEK293 cells with two genes X and Y. Gene X is tagged with Myc and Gene Y is tagged with GFP. The transfection map is shown below with controls:
1st well - GeneX-Myc
2nd well - GeneX-Myc+GeneY-GFP
3rd well - GeneX-Myc + GFP
4th well - GeneY-GFP
5th well - GFP
After lysing the cells, the proteins were pulled down using GFP magnetic beads. We observed bands for GeneX-Myc in the 2nd well (Great result!!!) but surprisingly we also observed less intense band specific for GeneX-Myc in the 1st well too on our western blot. No cross contamination or mix up of samples were observed in our analysis. For your reference, I am attaching copy of the blots. We have repeated the experiment twice. Any explanations!!!
I agree with Spanou to an extent, but if going by Spanou's explaination, you should also see a faint band in well 3 also, which you do not see. Was there any chance that your sample from well 2 spilled into well 1.
I don't agree with Rakesh because EGFP alone always express better that fusion form and also it's better recognized by anti GFP due to small conformational changes that happens during fusion protein expression. I believe that when you use EGPF alone it binds completely to your bead and blocks the minor cross interaction with Myc (what doesn't happen when you have only Myc).
1. Are you using reducing agents in your lysis buffer (DTT)?
2. Which lysis buffer are you using? Is the salt concentration correct? Increasing salt concentration can help you with nonspecific background.
3. Are you blocking your beads with lysis buffer + 1% BSA before immunoprecipitation?
This is a common problem in immunoprecipitaiton, from the looks of it you'll need to increase either the number of washes or the stringency of them. I've never tried blocking beads, but you can pre-clear your lysates of things that stick to the beads, but this can be an issue if your protein of interest sticks to them...I generally do 5 x 5 min washes with 1 ml of wash buffer. I also like doing a salt gradient when optimizing an IP protocol, usually from 250 mM to 1000 mM to see my contaminants go down, leaving only the specific interactions. Good luck :)
@ Rakesh: I tested the blot with GFP antibody to check if the lysate is spilled into well 1. We were not able to detect any signal for GFP at all in 1st well.
@Andre: Thank you very much for your comments and suggestions. We would like to give a try with your blocking suggestion. Would it be good to block the beads with lysis buffer with 1-2% BSA for a period of 10minutes and wash once or twice with PBS. Looking forward for your comments. The composition of lysis buffer is as follows: (50mM Tris-HCl pH7.5, 150mM NaCl and 0.05% NP-40 with proteinase and phosphatase inhibitors). I hope the salt concentration is good enough for lysis and to avoid non-specific background.
Dear Chandra
You should check your bead data sheet for proper blocking time. I've used anti FLAG magnetic beads and I always blocked for 1h using 1% BSA. I don't think you should wash with only PBS because this will remove detergent from bead surface altering the detergent concentration when you add your sample. I always washed one time with lysis buffer. Ideally, as Sean stated, you could titrate the salt concentration. 150mM is standard but some cases require harsher conditions.
As others already mentioned, the myc-tagged protein may be interacting weakly (hence not seen in the presence of GFP) with your GFP antibody beads. This can be reduced by increasing salt concentration in the lysis buffer and washes of the beads. However, if you still see the band in GeneX-myc alone loaded lane, then you may want to change your GFP antibody used for IP or pre-treat your lysates with beads coupled with isotype control IgG.
If you want to be sure that your anti-GFP antibody is not recognizing your GeneX-myc, try a western blot with it, or a immunofluorescence/ELISA. You can also use a blocking-peptide for the antibody to ensure it is not background. There are several antibodies against classical GFPs, unless you're using the copepod one. You can try with another recognizing a different epitope from GFP.
Indeed, affinity for GFP is clearly higher than for your protein, therefore, lane 3 is a good negative control.
You can co-transfect different amounts/ratio of geneY-GFP:GFP and same geneX-myc and see that geneX is recovered better when this ratio increases. As an internal control for your experiment.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA