I am trying to pulldown a protein of 300 kDa using protein G agarose beads. I used two negative controls, one pulldown with just beads (without primary antibody) and another pulldown with another non-specific primary antibody. I used rabbit primary antibody for pulldown and mouse primary antibody for detection by Western blot. A band at 300 kDa was detected in pulldown sample with specific antibody, total cell lysate and pulldown sample with only beads. However, when I probed for another protein of 43 kDa (that might be interacting with the 300 kDa protein), a band at 54 kD (band density more than that of 300 kDa protein) was seen in the pulldown sample with non-specific primary antibody, total cell lysate, pulldown sample with specific antibody and pulldown sample with only beads. Is it due to the nonspecific binding of the protein (54 kDa) to the beads or the primary antibody is interacting nonspecifically to the unknown protein of 54 kDa? If it is due to the nonspecificity then how can I avoid it.
Here is the work flow of my Co-IP experiment:
1. Pre-clearing of lysate: 500 ug of cell lysate was mixed with 40 ul of 50% bead slurry and incubated for 1h at 4° C. Later, the mixture was centrifuged and the supernatant was collected for pulldown.
2. Antibody was added to the collected supernatant and overnight incubated at 4° C.
3. 40ul of 50% bead slurry was added to the antibody-protein mixture and further incubated for 1h at 4° C.
4. The beads were washed three times and 20ul of 2x laemmli buffer was added and heated for 10 minutes at 100° C. Western blot was done.
You can easily work this around by performing diafiltration of the samples on an 100kDa Amicon ultra filter, prior to performing your pulldown assay. Everything under that molecular weight will go away. Also, if membrane clogging occurs, you appeal to Mg2SO4 protein precipitación to further separate the proteins in the solution in several fractions.
Hasan Issa, I am using mouse IgG for pulldown and rabbit IgG for western. How come the mouse IgG heavy chains be detected by anti-rabbit secondary antibody? These bands were present in the total cell lysate as well.
@Mukund P S
Thank you for your valuable answer. Next time I will check with the conformation specific secondary antibody.
Are you sure of the sizes of your target protein, 43kDa. Is it possible that the 54Kda protein is the same protein but glycosylated or with some other modification. Not sure what lysates you are using, mammalian/Ecoli?
Could try
1. Blocking beads with BSA
2. Pre bind antibody to beads then add to lysate
3. Pre-clear with beads + irrelevant antibody
4. Increase stringency of buffers for pulldown
5. Test for cross reactivity in WB: run Rbt ab on WB and probe with mouse: Use reducing and non reducing conditions.
@ Ian R Wilkinson,
I am using mammalian cell lysate. My protein of interest is cytoplasmic protein. The predicted molecular weight is 42 kDa.
1. Yes, as you suggested I can try blocking the beads.
2. Can you please suggest me a good protocol to pre-bind antibody to the beads.
3. I did pre-cleared the lysate but the yeild was very less.
4 and 5. I will try them as well.
The easiest way to pre bind ab is to simply mix beads with Ab at saturating concentrations. However, if your Ab is expensive this is really not a good idea!! The other way is just bind in the ratio that you use in the experiment, for example 25 uL beads plus 1 microg Ab etc (Scale up as necessary). Check supernatant for Ab, it should all be captured, wash beads, then block in BSA, wash again. Try a range of buffers with different detergents. It is worth spending a little bit of time reducing the non-specific binding to beads prior to doing a full experiment and using up primary Ab.
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