Hi scholars,
I am planning to conduct western blot to detect MUC2 protein , which estimate have about 540kDA size.
Judging by this huge size, I wonder can the protein be electrophoresised and transferred into the blot? Can someone suggest a western blot protocol for this huge protein?
This molecule is huge in size.. May u ask why don't you perform ELISA for this one
Hi,
You definitely should try Tris-acetate gels from either Invitrogen or Bio-Rad.
I curious, how do you plan to express it?
To answer you're question, you might want to lower your gel percentage to allow for bigger pores. A poster at protocol online has been successful using a 5% gel. See her full recipe and protocol below.
http://www.protocol-online.org/biology-forums/posts/29214.html
Hi,
You are lucky to work with such a big protein. Its really challenging to work with it. MUC2 mwt in SDS-PAGE gel is more than calculated because of glycosilations and proline richness. Its 3day process. Just prepare 6% SDS-PAGE gel(stacking gel itself is 5%).You should take care at the boiling step where you should allow BME in sample buffer do its job well(5-10min). Run the gel in 80V initially followed by 100-150V but keep cold conditions for longer time(better have high molecular weight ladder >250kd). After passing all the bands from gel up to 110kD,go for wet transfer(gel will be more viscous, so open casset in MQ(buffer is slippery makes gel tear apart) filled water tank slowly then incubate in transfer buffer with increase SDS concentration to 0.1%. 20V for >16hr in 4degrees, if you use methanol activated PVDF, no need to add methanol to transfer buffer). Incubate the blot for MUC2 {and for loading control run the other gel with high percentage with same amount of protein}. All the best.
You can try agarose sds gel that are suitable for hmw proteins. Blotting protein from agarose to membrane are 15% more efficient.
Dear Mr. Wai chin chong
You can try 4-15% gradient SDS-PAGE and tris -acetate gels are most suitable for your experiment try to fabricate your own resolving mesh as that would be cost effective.I'd like to suggest you that you to try analysing you subject at transcription level or try developing ELISA for for your protien of interest if you antibody is ELISA compatible.
We use 3-8% nupage gels, tris-acetate buffer for a 250kD protein with an overnight transfer. our ladder goes up to 450 and it all transfers
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