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Western for very small protein


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#1 wincel

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Posted 01 February 2011 - 02:03 PM

I'm trying to detect if a so called ribozyme-skipping sequence (RSS) between two proteins is effective. I have a flag-plus-a-little-extra dummy protein first with start codon, the RSS and afterwards (without start codon) GFP. I can see GFP under the microscope readily. In Western I did 10ug and 60ug and I can see tubulin nicely as standard. With flag I see a huge band at the potential fusion protein of flag-dummy-GFP (30kDa) and a strong band a bit below (12kDa) but nothing where I would expect the flag-dummy only (4.3kDa).
We did a 15% SDS-PAGE and used a Hybond P PVDF membrane with pore size 0.45micrometer and have done a wet transfer for one hour at 100V. Is it possible the flag-dummy protein just went through the membrane? We can see the 10kDa band of the ladder readily.

#2 BioMiha

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Posted 01 February 2011 - 10:56 PM

I would suggest a Schaegger (Tris Tricine) PAGE. With that you might just be able to see your 4.3 kDa protein. Using 10-60 ug on a western is a bit overkill don't you think? Or is that whole protein?

#3 wincel

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Posted 02 February 2011 - 11:04 AM

That amount is whole protein. What does Tricine do?

#4 BioMiha

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Posted 02 February 2011 - 11:06 PM

Tricine replaces glycine in the running buffer. If I remember correctly the small highly hydrophobic peptides get trapped in micelles formed by SDS in the running buffer. So, by using a lower SDS content and Tricine these micelles are not formed (or are formed differently, who knows), therefore you can resolve lower Mw peptides. Works down to about 5 kDa, supposedly less, however I haven't been able to resolve really small peptides myself.




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