Hello scientific world!
I would appreciate your insight/experiences with the following:
Has anyone else observed that polyphenols that have been complexed with proteins can (after SDS-PAGE) be transferred with the proteins onto a membrane (I use PVDF) and create 'ghost bands' or 'white areas'? I highly suspect that this can happen and that ghost or white bands/areas appear because the presence of polyphenols is interfering with any subsequent immunoblotting steps (such as the initial binding of the primary antibody) so no signal will be created at the end.
Disregard the lane with *
Thanks much in advance!
Best
Nat
How did you confirm that polyphenols are attached to polypeptides or monomeric proteins (protein bands in SDS polyacrylamide gel following SDS electrophoresis) ? If polyphenols are complexed with proteins, what sort of interactions (covalent, ionic or ..) are you expecting between them? Can polyphenols remain in association with proteins following SDS treatment and subsequent processing? (of course, polyphenols would remain in association with proteins provided they have covalent interaction)
Did you use PVP or PVPP for removing phenolics or polyphenols?
After seeing your blotting membrane it seems there is a kind of handling problem... In this blotting membrane even the protein ladder is not visible clearly though I can see some band on it. I think more attention is needed while estimating the protein concentration, loading the sample and transferring, regarding to unspecific band, if you use polyclonal antibody it is obvious and I believe occurrence of unspecific band would not be the problem if you get the specific band on the desired location..
Dr. Pardha-Saradhi,
Polyphenols usually interact with proteins via non-covalent forces such as hydrophobic interactions and hydrogen bonds, but you can also find in the literature that, under certain circumstances, they interact covalently. In fact, this is something I still plan on investigating for my research samples. I know for example that NBT (nitroblue tetrazelium) has been used before (e.g. Hagerman et al) to stain quinone-containing complexes to show that polyphenols were covalently bound to proteins. Based on other experiments I've conducted I would not necessarily exclude the possibility of covalent linkages. Also, protein-polyphenol complexes have shown to be able to appear as smears on SDS-PAGE, which I have seen before and what can also be found in the literature (to better show my samples I've also attached the associated SDS-PAGE).
As for your question, if polyphenols can remain in association with proteins following SDS treatment and subsequent processing: I don't know. That's what I am wondering about as well. My experiments so far highly indicate that they do, at least partially. SDS would break up non-covalent forces, however, after denaturation polyphenols still remain in the sample solution, so they may be able (if it wasn't covalent) to partially remain associated or re-associate before sample loading? (which is shown by smears on the gel)
No I have not used PVP or PVPP to remove polyphenols. I have come across these chemicals before when reading into the literature and would have to look it up, if it would be useful in my experiments.
Chand,
I might have to give more information. The marker for Western blotting I am using is in fact not the best (some higher molecular weight bands for some reason usually don't show up) but in this case this was a preliminary experiment (not for publication) and I only used a few uL of the standard (what I had left) instead of 10 uL what I usually use, that's why the bands rather appear more faint, but I could still detect standard bands up to 50 kDa. (see the new pic attached to Dr. Pardha-Saradhi's answer). Protein content in all samples was measured using a nitrogen analyzer in duplicate with pretty much no standard deviation (it's the best protein content estimation one can get). When you read my answer to Dr. P you see that protein-polyphenol complexes can be seen as a smear on a gel, and if you take a close look at the figure you can see that with increasing polyphenol content in the region where white spots appear on the western blot,increased smears/darker it gets in the same molecular weight region on the gel.
And as you can see, the control, which does not contain polyphenols but is just the protein itself, does not show white areas/ghost bands.
The * sample does also contain a lot of polyphenols but the original sample preparation was slightly different compared to the other samples containing polyphenols.
Since equal amount of protein was loaded, I wouldn't think that ghost bands appeared because of too much protein loaded, or too much antibody...if that were the case I would have seen it for the control as well. there is clearly a polyphenol concentration effect. And, as you can see on the gel, in that MW region where a lot of the white area increases on the Western, there is no specific band on the gel, but rather a smear that increases in intensity with an increase of polyphenol concentration, if there weren't any target proteins, I should get a similar grey background on the western blot as I can see for the control (I am blocking with BSA, so sometimes I do get somewhat a more or less grey background).
I hope this helps.
Thanks
Come to find out that proteins were transferred across all lanes and that polyphenols present on the membrane likely either a) increase binding to secondary AB/enzyme complex molecules (therefore high concentrations of secondary AB --> rapid depletion of substrate in those areas --> white ghost bands/ burn out of light) or b) contribute to substrate depletion by non-enzymatic conversion of luminol to its oxidized form with the same result. If anyone has made similar experience please let me know. In the process of testing this hypothesis.
- Badges
- Science method