Q about transfer - (Mar/17/2008 )

I used transfer buffer which prepared 5 days ago, to transfer protein to NC menbrane, but failed.
due to de buffer?

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Hi,

If buffer prepared correctly, shouldn't be a problem.Try manipulate the Amp and time(100mAmp,2hrs..maybe).The outcome also depends on the protein size.Larger size will transfer slower.Good luck.

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What's in your transfer buffer? I always add my methanol fresh, so old transfer buffer with MeOH may be a problem.

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Thanks! I've heard that protein will pass through membrane during a long time transfering. This time, I spent 80 min, but still got no signal after blot.

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My buffer:
45% dw
45% methanol
10% Acetic acid

Maybe.
I tried new prepared buffer this time, but still, failed~
My protein was extracted from yeast. May it be a problem?

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You could try a different transfer buffer. This is my recipe:

10X Transfer Buffer (1L)

Glycine 75.07g
Tris 12.11g
Add dWater to 1L.

To make 1X: Add 100ml 10X buffer to 200ml methanol and make up to 1L with water. Store at 4deg, use the same day.

For the transfer: 70 mins at 100V in the cold room (don't forget your magnetic stirrer!).

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[/quote]

You could try a different transfer buffer. This is my recipe:

10X Transfer Buffer (1L)

Glycine 75.07g
Tris 12.11g
Add dWater to 1L.

To make 1X: Add 100ml 10X buffer to 200ml methanol and make up to 1L with water. Store at 4deg, use the same day.

For the transfer: 70 mins at 100V in the cold room (don't forget your magnetic stirrer!).
[/quote]

Oh~ I made some mistakes. That's not de transfer buffer, it's destain buffer~~
well, thank you. I'll try~

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