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protein not stained by Coomassie Blue


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#1 rock&science

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Posted 15 October 2012 - 06:17 PM

Hi guys, just wondering if you all know whether it's possible that Coomassie Blue cannot stain certain protein.

#2 bob1

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Posted 15 October 2012 - 06:54 PM

Basically no, but there are detection limits...

#3 rock&science

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Posted 15 October 2012 - 07:34 PM

thought so. But recently, I loaded about 30micrograms protein (estimation based on A280), but did not see any band on my Coomassie.

#4 bob1

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Posted 16 October 2012 - 03:15 PM

Purified protein? cell lysate (from what)?, size?

Did you try another quantitation method, there are heaps of things that absorb strongly at 280 nm, including DNA, RNA , phenolics, sugars....

#5 charleyhorse

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Posted 17 October 2012 - 01:05 PM

Indeed it is possible. Coomassie blue only stains arginine and aromatic amino acids so your low protein concentration in conjunction with the possibility that your protein may be low in these particular amino acids, it very well could stain poorly or not at all.

#6 DRT

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Posted 18 October 2012 - 11:04 AM

Indeed it is possible. Coomassie blue only stains arginine and aromatic amino acids so your low protein concentration in conjunction with the possibility that your protein may be low in these particular amino acids, it very well could stain poorly or not at all.


I think it would be more correct to say that coomassie blue has a preference for arginine (and basic aa in general) and aromatic amino acids.
Even poyglutimate is able to show some coomassie binding, but admittedly this could be due to the charge on the N-terminal.

#7 rock&science

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Posted 21 October 2012 - 03:24 PM

thanks for the replies guys. It's a purified protein (from affinity column) which has been dialysed and partially freeze-dried. Is it possible that glycosylation/mannosylation affected the staining?

The other thing is, I recently loaded ~10micrograms for silver stain, and it stained quite weakly - so I knew that the concentration from A280 is a bit overestimated. But here's the kicker, I loaded 20x more than I loaded in Silver stain for Coomassie Blue, and I still got nothing.

Edited by rock&science, 21 October 2012 - 03:25 PM.


#8 mdfenko

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Posted 22 October 2012 - 07:33 AM

thanks for the replies guys. It's a purified protein (from affinity column) which has been dialysed and partially freeze-dried. Is it possible that glycosylation/mannosylation affected the staining?

it could but it's more likely diffuse

The other thing is, I recently loaded ~10micrograms for silver stain, and it stained quite weakly - so I knew that the concentration from A280 is a bit overestimated. But here's the kicker, I loaded 20x more than I loaded in Silver stain for Coomassie Blue, and I still got nothing.

not necessarily overestimated. we've seen proteins that don't show up well until stained with silver for a second time (with or without destaining between stainings).

are you using coomassie g or r-250?

g is more specific than r so you may get stronger staining with r.

Edited by mdfenko, 22 October 2012 - 07:34 AM.

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#9 Marjolein

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Posted 16 November 2012 - 02:16 AM

Hi all,

I had quite a strange result yesterday. I tried to stain purified proteins in an SDS gel with Coomassie (G-250). I have done this experiment earlier, and then I had no problems at all. This time I used the same proteins, the same gel, the same Coomassie, but one of the proteins does not stain, though I am sure that the protein is there. Concentrations are sky-high.

I tried O/N staining with a fresh Coomassie solution but it didnt help. I also put some dilutions on gel (since the concentration is so high) to see if this could help, but again no effect.
The strangest thing is, usually this protein stains very well under these conditions with Coomassie. However, with silver staining it always gives negative staining, regardless the concentration.

Now I am running out of ideas, does any of you have a clue?

Thanks,
Marjolein

#10 mdfenko

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Posted 16 November 2012 - 09:41 AM

are you sure you didn't use coomassie blue r-250 when it worked? r-250 is less specific than g-250 so it stains more.

as for negative silver staining, we had proteins which exhibited this phenomenon. we found that staining twice with silver would make the band show up (with or without destaining in between).
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#11 Marjolein

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Posted 20 November 2012 - 05:21 AM

Yes, I'm sure I have used the same bottle of Coomassie (and I have never used R-250 before, so there is no bottle R-250 stock on my lab table).
My colleague found out that cobalt ions might interfere with Coomassie staining, so maybe some cobalt eluted from the column with my protein.

The protein did stain well on nitrocellulose with Ponceau, though. At least we are sure enough now that the protein is indeed present.

Thanks for your tip on the silver staining, I will try it!

#12 Marjolein

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Posted 20 November 2012 - 05:27 AM

So what did you do exactly with this silver staining? Did you repeat all steps including fixing?

1. Fix in 50% ethanol / 10% acetic acid
2. Fix in 30% ethanol
3. Sensitize
4. Stain
5. Develop
6. Stop

(I used the Fermentas PageSilver kit for silver staining)

#13 mdfenko

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Posted 20 November 2012 - 12:15 PM

we used a modified (by us) method of merril, et al (science, 211, 1437-1438 (1981)).

whether we destained first or not, we start restaining from the sensitization step. the proteins are already fixed and the sds is already removed (ethanol step).
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#14 Marjolein

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Posted 22 November 2012 - 01:27 AM

Ok, thanks a lot! I will let you know if it helps. Posted Image




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