We have come across a strange issue with a western blot optimisation.
We are probing for Akt (p). We keep (done multiple times, because I thought it was operator error) getting completely blank membrane, no ladder even! We are using tris glycine gels, magic marker on the gels.
The confusing part is that we have also run the gel, blotted then split the membrane to run two different primaries (same secondary and ECL reagents). The results of the other primary look good, we can see the ladder etc, but the pAkt has nothing, not even ladder. We have even tried overnight low voltage blotting in case we aren’t getting complete protein transfer, although it doesn’t explain why the ladder is missing in the pAkt blot and not the other. We can try increasing the protein loading, but still dosent explain the missing ladder.
This is especially confusing as my technician had this assay working (using Tris acetate) gels a couple of years ago. …and yes we have bought fresh antibody and tried old and new.
Stuck.
Hi!!!
have you stained the membranes with Ponceau? if you are getting protein, but no blot at the end, you can start looking for less membrane blocking, change milf for BSA or viceversa, higher Ab concentration, another Ab brand and so on. I did not know that the Magic Marker could interfere with some Ab, but that's a good piece of info... please let us know!!! Good luck!!
Bea
i thought your Membrane is not compatible with your transfer buffer, i also suffered with this problem, it was resolved after changing my transfer buffer.
Hi Kate,
if you are able to detect ladder and bands for other proteins there is very less probability that your gel or transfer method has an issue.
can you please tell few more details?
1. what is the source of primary and secondary antibodies that you use to probe pAKT (goat, sheep, rabbit, mouse etc)
2. is it the same secondary antibody that you used for probing the other membrane (protein)?
3. what blocking method did you use?
Apparently you have problems for electroblotting, have you stained the membrane after transference?
Removing the Marker did not alter results. To answer some of the questions above. I am using PVDF membrane and tris-glycine gels and TG transfer buffer with 20% methanol. The primary is rabbit polyclonal, the secondary is goat anti rabbit and we have been using the same secondary for several other western protocols with no problem. I use 5% BSA blocking. I have stained the gel after transfer and see some protein remaining, but not much. I havent stained the membrane as my other blots have been working fine at the same conditions, same gel chemistry and similar size range. I wasnt using reduced conditions as it wasnt mentioned in previous notes from other lab member, but will test that next, although it still dosent explain the lack of Magic Mark TM on the blots done for this particular experiment.... Thanks for all your comments.
So except the pAKT primary antibody everything else seem to work. May be you can load the pAKT primary antibody, run a gel along with a ladder and transfer to see if you are able to visualise the heavy and light chains of the antibody. I am not excluding the possibility of a contaminated antibody.
You can try your first antibody activity by dot blots or double immunodifussion in agarose or agar.
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