Transfer during western blotting - (Jun/06/2007 )

Hi all,
yesterday i did a western and my mentor said that the transfer was not good. he told me to calibrate the conditions. So, maybe someone will help me with some virtual calibration.
the things is that i need to see a 80kDa protein that is glucosylated and reaches 140kDa. so on western it will be 140kDa. because of it weight and moreover because of the sugars it is probably difficult for it to go out of gel into the membrane. Is anyone has experience with hight weight and glycosulated proteins, what should be the transfer conditions? any other suggestions are highly encouraged.

10X

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After running the SDS-PAGE, do not soak the gel in transfer buffer for a while to get rid of SDS because SDS assists the transfer of large MW protein onto the membrane.
In addition, you may need to use the tank transfer rather than the semi-dry one.

Hope this may help.

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search the forum for this topic. I think there were other posts related to this topic.

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deglycosylate if the glycosyls not interesting for you, or use O.N. tank blotting (constant 220-230 V for 16 h under cooling)

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Hi,

It would be easier to answer your question if we knew how you do the transfers?
Is it a wet transfer? What voltage do you use? Do you ponceau? Do you have a positive control?

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hi again, thanks for your replies till now.
is it possible to get rid of glycosylation after collecting the protein? the over nght blotting sound interesting, but isnt the voltage too high?
the exact conditions of the western were:
running in 8% acryl amide gel for about 1.5h
weting the membrane (nitrocellulose) in transfer buffer (80 running:20 methanol) fro about 20 min
wetting the gel in transfer buffer for about 20min
wet transfer 80V (equivalent to 0.15mA) for 1.5h in ice

controls: prestained weight marker 30-170 kDa weight range. I check that it is transfered to the membrane. pauncheo S staining of the mebrane to assure transfer and in some cases staining the gel with coomassie blue.
as for the antibody, it should work because other people used the same antibody and got good bands.

so, what and where is the problem??

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You can deglycosylate the protein using deglycosylation enzyme. I transferred overnight with constant 100mA in the cold room. It was ok. You can also try to modify your transfer buffer by adding the SDS to increase the efficiency.
Hope it helps.

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you can deglycosylate even on Western blots but use both N- and O-glycosidases;

voltage as indicated works fine; current/voltage is much higher for tank than semi-dry blotting

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I use 80:20 transfer buffer:methanol as the final transfer buffer and wet the membrane as the gel is running. However, I do not wet the gel in transfer buffer. I transfer for 2 hours at 250 mA on ice and my protein is 66 kDa. I don't have experience with glycosylated proteins but if it is 140 kDa, you may want to transfer for 2.5-3 hours since it takes longer for larger proteins to transfer. Hope this helps!

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