in-gel digestion from unstained bands

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pdow01
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in-gel digestion from unstained bands

Postby pdow01 » Fri Jun 13, 2014 7:37 am

Hi All,

I want to run my samples in 2 lanes on a 1D gel with molecular weight markers beside both lanes, 1 lane of sample and a lane of molecular weight markers will be silver stained and I will use this as a reference to cut from the other lane of sample and molecular weight markers that are unstained. Do I need to fix the unstained lanes also? Any other comments would be helpful. The unstained gel slices would then be subject to in-gel digestion for MS. levels of my protein of interest are likely to be very small that is why I don't want to stain/destain

Regards,

Paul.

Infinity
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Postby Infinity » Fri Jun 13, 2014 11:07 am

You have to fix otherwise your protein bands will spread in all directions. I'm not sure why it is not possible to in-gel digest after silver-staining.

gadsouza
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Postby gadsouza » Mon Jun 16, 2014 4:22 am

Probably PMID: 17326674 is a good reference for you to get those tips

botte
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Why not try Coomassie?

Postby botte » Tue Jun 17, 2014 2:23 am

Hi Paul,
if you do not expect to see staining anyway and just want to visualize the MW markers, why not go for Coomassie staining? A bit easier than Silver and definitely MS-compatible. However sames as Infinity, I would also expect Silver Staining to work with MS detection if done right.
Christof

gadsouza
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Postby gadsouza » Tue Jun 17, 2014 5:24 am

I am supposing his concern is to avoid too many band washes to destain the bands, and therefore decrease sample loss.

m/z
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Postby m/z » Tue Jun 24, 2014 11:48 pm

@gadsouza:
But is there any evidence that (prolonged) washing/destaining of gel-peices can result in loss of proteins? My impression was that proteins do not easily escape the gel matrix?

gadsouza
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Postby gadsouza » Wed Jun 25, 2014 2:58 am

In the paper I gave the PMID above, they showed that loss is quite high for LMW proteins (below 20kDa) (Figure 3A).

m/z
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Postby m/z » Wed Jun 25, 2014 3:13 am

Thanks! Interesting...

m/z
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Postby m/z » Wed Jun 25, 2014 10:23 pm

@gadsouza,
This is an interesting paper (by top researchers in the field). They show that peptides (and actually also proteins>20kDa) can passively elute from TRICIN gels and, secondly, that passive diffusion happens with treatment with acetic acid, etc. However, I assume that most gel-lcms workflows use SDS-PAGE and fast comassie staining (no fixing step). Still from this paper it appears that loss of <20kDa proteins could be a substantial limitation of gel-LCMS as compared to for example MudPIT or RP&LCMS?
I think the common assumption is that you can leave gel peices in the fridge for months and still get good ID with in-gel digestion - at least that has been my experience. But maybe I am wrong?
I have just started an small experiment to compare commasise staining and destaining vs. no staining to see the impact on the identification of low mass proteins. I expect that in particular the destaining step could be the source of some sample loss?


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