[Forsh]

Hey,

Over the last few weeks i've been running some experiments on protein expression which has involved running cell lysate through SDS-PAGE and then using Western blotting techniques to probe for a certain protein that we're looking for.

So far I've observed over and over again a double banding of the protein on the Western blot. I've been running reduced gels, so it's not a dimerisation; besides, the molecular weight of the protein is around 30 kDa and the bands are about 5 kDa apart. It's definitely not a cross reactivity either. The current hypothesis is that there is a conjugate molecule attaching itself to some of the proteins - I have been treating the cells with an agent to induce oxidative stress, which could result in S-glutathiolation in a dose dependent manner (more stress = more glutathiolation = a higher number of the proteins becoming conjugated). This conjugate molecule isn't so much adding weight to the protein, as reducing the protein's motility through the gel, making it appear lighter as it travels less distance.

With this in mind I was wondering, does anyone know any papers which describe protein conjugation causing this kind of effect through a decreased motility mechanism? I can't find anything anywhere! Also, does anyone have any other suggestions about what could be causing the double banding? The experiment was repeated several times and i'm working with (and being supervised by) a PhD student who taught me the method and has been refining it for years, so we're pretty sure it isn't an experimental error.

Thanks!

:EDIT: Attached image shows the double bands. The lanes are: Control, 4 of increasing treatment concentration, then a protein positive sample (I know I overloaded it in this image, its just the best one I have at hand). I've turned the contrast up a bit and it looks like the control has a slight double band, but a third band is beginning to appear at the higher treatment concentrations, which I didn't notice before. The matching GAPDH blot appears fairly evenly loaded, though I haven't conducted densitometry analysis on it.

Attached thumbnail(s)

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This post has been edited by Forsh: 8 February 2012 - 07:36 PM

[Max_Normal]

Looks like a massive shift just to be down the the charge on the glutathionine doesn't it? From what I remember, this just gains or loses a single proton, so the shift would be relatively small. If the protein was undergoing multiple glutathiolation (or phosphorylation), you'd perhaps to see more of a ladder of differently migrating bands. Perhaps that's what the third band shows, the difference between zero, single or double glutathiolation?

Are you certain that this is not a splice variant or isoform that is being differentially expressed as a result of your treatment?

Have you considered sumolation and ubiquitination? Your band shift is not far off the size of ubiquitin, and this is a covalent modification that is resistant to reducing conditions.

This post has been edited by Max_Normal: 3 March 2012 - 03:53 PM

[Forsh]

Max_Normal, on 3 March 2012 - 03:20 PM, said:

Looks like a massive shift just to be down the the charge on the glutathionine doesn't it? From what I remember, this just gains or loses a single proton, so the shift would be relatively small. If the protein was undergoing multiple glutathiolation (or phosphorylation), you'd perhaps to see more of a ladder of differently migrating bands. Perhaps that's what the third band shows, the difference between zero, single or double glutathiolation?

Are you certain that this is not a splice variant or isoform that is being differentially expressed as a result of your treatment?

Have you considered sumolation and ubiquitination? Your band shift is not far off the size of ubiquitin, and this is a covalent modification that is resistant to reducing conditions.

Max, you are brilliant!! Ubiquitination fit the bill perfectly, I don't know why I overlooked it. Densitometry analysis shows that as treatment conc increases the heavier band increases, the lighter band decreases, and the overall intensity of the bands decreases - obviously because my protein is being degraded. It could also kind of explain the middle band, it could be that the protein has been cut and the antibody is binding to a distinct fragment. Though I don't know too much detail about ubiquitination so I will have to read up on this to see how viable that explanation is. I'm not sure if it is more likely that i'd get a low MW smear.

I actually took a closer look at my blots the day before you posted this and decided the weight difference was more like 8-9 kDa, which fits (the lighter band is my protein and the heavier is the unknown variation). There are 4 (or 6, I don't have my papers to hand) sites for glutathiolation on my protein, so I think there would be more bands in between showing varying states of glutathiolation.

I'm fairly confident that i'm not looking at splice variants because all of the variants are lighter and i'm only seeing a heavier unknown.

My protein does undergo glutathiolation and phosphorylation regulation, so I will have to spend some time considering each possibility, but ubiquitination would make a lot of sense.

Thank you!

[Max_Normal]

Forsh, on 3 March 2012 - 05:10 PM, said:

Max, you are brilliant!! Ubiquitination fit the bill perfectly, I don't know why I overlooked it. Densitometry analysis shows that as treatment conc increases the heavier band increases, the lighter band decreases, and the overall intensity of the bands decreases - obviously because my protein is being degraded. It could also kind of explain the middle band, it could be that the protein has been cut and the antibody is binding to a distinct fragment. Though I don't know too much detail about ubiquitination so I will have to read up on this to see how viable that explanation is. I'm not sure if it is more likely that i'd get a low MW smear.

I actually took a closer look at my blots the day before you posted this and decided the weight difference was more like 8-9 kDa, which fits (the lighter band is my protein and the heavier is the unknown variation). There are 4 (or 6, I don't have my papers to hand) sites for glutathiolation on my protein, so I think there would be more bands in between showing varying states of glutathiolation.

I'm fairly confident that i'm not looking at splice variants because all of the variants are lighter and i'm only seeing a heavier unknown.

My protein does undergo glutathiolation and phosphorylation regulation, so I will have to spend some time considering each possibility, but ubiquitination would make a lot of sense.

Thank you!

2 easy ways to test this, obviously an antibody to ubiquitin, but if you treat with the proteasome inhibitor mg132, you might see a build up in intensity of your heavier band along with the disappearance of the third band if it is a degredation product.