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Marjolein

Member Since 16 Nov 2012
Offline Last Active Jan 08 2013 03:15 AM

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Active Posts 6
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Age 25 years old
Birthday February 11, 1988
Gender
Female
Location
Amsterdam

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My research interests
Molecular microbiology, protein-protein interactions, pharmaceutical sciences, medicinal chemistry, chemical synthesis

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In Topic: Premixing gel solutions

27 November 2012 - 06:15 AM

Ok, it is clear now:

I tested it with a small amount (for appr. 10 gels) but within a week my gels already started to get fuzzy!
And even worse: some of my proteins traveled even faster than the dye front, so they ran off the gel.

So gels should really be freshly prepared!

Thanks for your reply.

In Topic: protein not stained by Coomassie Blue

22 November 2012 - 01:27 AM

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

In Topic: protein not stained by Coomassie Blue

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)

In Topic: protein not stained by Coomassie Blue

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!

In Topic: protein not stained by Coomassie Blue

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

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