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mhneupane
Joined: 19 Feb 2009
Posts: 1
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 Posted: 19 Feb 2009 9:14 pm
Post subject: low molecular weight protein
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Hi, i am working on stx2b protein on e. coli. my protein size is very small 10 kda. i couldnot detect it on western. i am using flag (M2) to detect it. what should be the precentage of sds gel and running time. i am using biorad mini protean cell for page and transfer. Moreover, i am worried about transfer time, right now i am doing 100V for 1 hour. can anybody help me. thanks in advance.
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AP
Joined: 27 Mar 2008
Posts: 3
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Posted: 21 Feb 2009 6:56 am
Post subject: low molecular weight protein
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A few suggestions:
For low molecular wt protein separations, you have a choice of gel percentages:
- 12% or 15% Tris-HCl gels
- use gradient gel 4-20%, 10-20% Tris-HCl gels, if you have higher mol wt proteins of interest as well
- Tris-Tricine gels are optimized for low molecular wt proteins (Ideal for peptides) separations. These gels require Tris/Tricine/SDS runing buffer.
For transfer:
- Use 0.22 um transfer membrane (NC or PVDF)
- Transfer buffer: no SDS, increase Methanol concentration upto 20%.
- Transfer at 200mA (constant current) for 30-45 mins.
Hope this helps.
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kathleend
Joined: 18 Mar 2008
Posts: 17
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Posted: 18 Mar 2009 1:58 pm
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try a 15% gel and make sure you transfer for a longer amount of time - between one and two hours.
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brichhardson
Joined: 27 Mar 2008
Posts: 9
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Posted: 14 Apr 2009 8:10 pm
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It may be too late to help you, but perhaps this can help someone else...
A 10 kDa protein is going to run at the dye front in either a 4-15 or 4-20% gradient gel.
I usually run proteins <15kDa on an 18% gel. Unfortunately, this results in a really long run time.
I can't help with the transfer.
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ranveerj
Joined: 04 May 2010
Posts: 3
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Posted: 04 May 2010 3:53 pm
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Hello Mhneupane,
I am working with core hsitones (12-17 KDa) and do a lot of SDS-PAGE and WB for the same. We use MP3 and transblot for transfer from BioRad. trying using 15 % gels for low molecular weight proteins. Run your gel and very slow current (<25 milli amps) and let it take longer time. Don't let the dye front run out. It should just reach the base of the gel. You can also use the dual color or kaleidoscope marker from BioRad for easy visualization of the migrating proteins.
Use wet transfer for transferring the proteins. I generally use 300 mAmp for 3-4 hours. You can also try the PVDF membrane specifically designed for low molecular weight proteins.
All the best!!!
Ranveer
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