Hi,
I want to carry out western blotting for a 233 kDa protein. I was told to use a tris-acetate gel due to the size of the protein. However, I was wandering would I be able to detect the protein if I use a bis-tris gel? As anyone tried this before? If so, what was your experience like?
Use 7 or 8% gel, and use 6M urea in your loading buffer - follow the normal wb protocol. It will work.
Dear Favour
as Mahesh suggest tris-acetate gel at 7-8% are the optimal for your size.
however if you do not have access to it, you can certanilly use a bis-tris 4-12% with new buffer. As the high mw band of markers as the novex sharp ( thermo lc5800) or biorad precision plus that are 250kda are able to enter in the gel and run on it, it theoretically will happen also for your protein. try to run the gel a lot ( i suggest at least 1h at 180V) and do the wb. it is possible that the transfer efficiency of a so big protein is not very high but if you need it for qualitative assessment it should work.
Good luck
Manuele
The larger the protein, the lower the acrylamide concentration (%T) needs to be to allow the protein to move in the gel. However, gels with less than 5% T are very soft and difficult to handle. You can avoid such problems by adding 0.5% agarose (electrophoresis grade) to the gel. The large pores of agarose allow the electrophoresis of whole virus, but the gel will much more stable. Just recall that the pores in agarose form during curing in the fridge for at least one night.
A Tris-acetat-tricine system (doi:10.1002/elps.1150040303, doi:10.1002/elps.1150040303) is suitable for proteins up to 500 kDa or so.
5%-6% (low percentage gel is hard to handle, you need to practice) SDS-PAGE gel, sample buffer using 1X SDS sample buffer (follow by cold spring harbor recipe). Gel running should be separated very efficiently ( only remaining above 180 kDa protein marker, your sample should be higher than this marker ). Transfer condition can be set by 0.5A (constant A) 60 min or 70 min (you must check the variable Voltage should not higher than 140V). Transfer buffer (25mM Tris, 192 mM glycine and 0.1% SDS including 15% Methanol which bought by sigma). The quality of Methanol should be very stringent, otherwise, your transfer efficient will not good ; at the same time, low percentage of 0.1% SDS will help transfer more efficient.
On the other hand, you need to check your 233 kDa target protein quantity in your cell type. If your cell type contains low protein expression, you should loading total protein more than 20mg for one lane, even you can load achieve to 50mg. Therefore, you can make the 15mm gel (10 wells comb) to enhance your total protein loading. Also, you can enhance your primary antibody titer or second antibody title, even use super ECL to improve your signal.
Hope these information may help you detect high molecular weight protein Western Blotting. I have been run more than 2000 thousands times for WB.
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