I am relatively new to conducting Western blots. I employed the alkaline lysis method to extract whole proteins from yeast. Subsequently, I introduced the Opti Protein Marker (ABM, CAT log NO: G252) and completed the gel electrophoresis. The proteins were then transferred to a nitrocellulose membrane using the wet transfer method. Following the transfer, I performed a Ponceau staining, during which the protein markers were clearly visible.
Moving forward, I proceeded to block the membrane for 1 hour in 5% BSA in TBST (Initially, I attempted blocking with 5% non-fat skimmed milk, but encountered high background signals). Subsequent to blocking, I washed the membrane with TBST (3 x 7 mins) and TBS (1 x 5 min). Following this, I incubated the membrane overnight at 4 degrees Celsius with a primary antibody (Beta-tubulin, Rabbit IgG Polyclonal), 1:1000 dilution in 5% BSA in TBST). Afterward, I repeated the washing steps with TBST (3 x 7 mins) and TBS (1 x 5 min).
For the next step, I incubated the membrane with a secondary antibody (Rabbit anti-Goat IgG (H+L), HRP, Polyclonal, 1:10,000 diluted with 5% non-fat skimmed milk in TBST) for 3 hours. Subsequently, I performed additional washes with TBST (3 x 7 mins) and TBS (1 x 5 min). Finally, I carried out chemiluminescence detection with an exposure time of 30 seconds.
However, my results were unsatisfactory as I observed multiple nonspecific bands, and the protein marker disappeared. I seek assistance from experienced researchers, especially those familiar with Western blotting of yeast proteins. I would appreciate any insights or suggestions to identify and resolve the issues.
Additionally, please refrain from suggesting the use of monoclonal antibodies, as it is currently beyond my budget constraints.
Thank you in advance for your help.
If you or others have not tested this antibody before then it's possible this is just a "bad" antibody that binds to multiple proteins. It's very common for antibodies to show different results than what is advertised by the company selling them.
I can't find much information about your protein marker, but you should make sure this marker is compatible with chemiluminescence. Ponceau staining will stain all proteins, so that's why you could see the marker protein there. But if the protein marker is not modified or your secondary antibody does not bind to the marker proteins, then you will get no chemiluminescence signal from the marker protein. You can read more about this at the bottom of this page: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/chemiluminescent-western-blotting.html
Also worth checking: when you say "Rabbit anti-goat" as your secondary, do you actually mean "goat anti-rabbit"?
Because the latter is what you need, and the former really won't work.
Secondly: why would your ladder necessarily appear via ECL? Unless every single protein in your ladder cross reacts with your primary or secondary, you won't see any bands via chemiluminescence. This is fine, since you have a distinctive blot shape with a snipped corner, and presumably used prestained markers, so you can always line it up with the images post-hoc. Or you can ponceau stain (or even coomassie) and take a picture of that for alignment. It doesn't need to be perfect, and size markers are always a little bit "ballpark" anyway.
Thirdly, do you have positive/negative controls? Because complaining about not having the budget for monoclonals isn't particularly relevant if you don't even have a way to confirm the antibody you DO have actually works, and recognises the target you're interested in.
Many antibodies (especially polyclonals) are quite backgroundy, in the case of polyclonals because they'll have all sorts of other antibodies the animal might have raised to other antigens. This is fine as long as your band of interest is clear and distinct, but without a positive control (and a negative)...how will you know? At present it doesn't matter how much your antibody costs, because you have no real evidence it works.
So...yeah, there's various optimisation steps that would be helpful to investigate here.
Dear colleague
It is very likely that it is due to the high specificity of your probe. The multiple bands must be fractions of your samples that contain some segment that hybridizes with your probe as well.
Thank you for your all suggestions Ciaran Daly John Hildyard Humberto Mejia . I'll try my best. I hope you don't mind if i come up with other doubts on my next trials.
If the ladder continues to wash away or fade you can (gently) mark next to each band with a pencil line. Put a small letter or number next to at least one band so you know what size it was. I used to use a ladder that had one orange/red band so I'd mark that one with an "o" to help keep track.
As others have mentioned, polyclonal antibodies can be messy. Hope you're getting better results.
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