Our lab purchased the pierce 10 min semidry transfer apparatus and it came with a free bottle of their 1-step transfer buffer (http://www.piercenet.com/product/pierce-1-step-transfer-buffer). That transfer buffer makes the 10 minute transfers possible, and it's a very high ionic strength buffer, and of course it's proprietary so the recipe is a secret. Instead of keeping buying the buffer, I want to make it our selves to save a little money. So far what I know is that the pH is 8.8, and it has more than 1.92 M Glycine, and 250 mM Tris (This is the 10X buffer that I had on my bench which is also pH 8.8). It does not appear to contain any SDS but it seems to contain something else in it like a certain type of alcohol that isn't methanol or else it would have to be listed on the MSDS. Anyone have any ideas what the missing ingredient or ingredients might be?
So far with my recipie of 1.92M Glycine, 250mM Tris and 10% Methanol, it transfers okay at 12-20 minutes with 18V and 1.3A per mini gel, but a little short at 10 minutes. The overall ionic strength of the homemade buffer seems to be about 20-40% weaker, and the test blots come out slightly blurry but still usable. I think the Glycine and Tris concentration needs to be higher and I need to use something else other than methanol.
The best semi-dry methodology I have come across is in an old paper (82?) by Khyse-Anderson. It's a three buffer system that works excellent and transfers at 300mA in 10-15 mins. I'll try to remember to post it in the morning.
We use the Trans Blot Turbo Transfer System from BioRad. We looked in the patent. And now we use the transfer buffer : 300mM glycine, 300mM Tris and 0.05% SDS. The transfer is o.k. in 10-15mins. The correct setting of the machine I have to check.
Do you have any updates as to which buffer worked best? We are looking into purchasing the Pierce Power Blotter system which requires the 1-Step Transfer Buffer. Thank you
336 mM Tris, 260 mM glycine, 140 mM tricine, 2.5 mM EDTA
works well for 7-10 mins transfer 1.2 A/25 V for 1 mini gel, or 10 mins 2.5 A/25V for 2 two mini gels
Anna Konovalova RE:
'336 mM Tris, 260 mM glycine, 140 mM tricine, 2.5 mM EDTA
works well for 7-10 mins transfer 1.2 A/25 V for 1 mini gel, or 10 mins 2.5 A/25V for 2 two mini gels'
is this recipe for 1x buffer?
has anyone tried 3min turbo transfer for 25-180kDa proteins maybe?
Thank you
Yes, it is 1x buffer.
I have not tried 3 mins transfer, but if you are using right thin (0.75 mm) gradient gels, it might work.
Good luck!
I don't know the secret of the 1step transfer buffer but here is a thing to try:
The recipe for the 1x transfer buffer is as follows:
29 g Tris Base
14 ,5 g Glycine
1 liter of absolute ethanol
Distilled water to 5 liters.
We tried to make a 2x transfer buffer and 7 minute transfer for a biorad minigel and it was not bad at all, except for the bands above 100 kDa.
I think that you should try to make a 3x transfer buffer solution, and you would be almost there. To avoid overheating it may be a good idea to use 2 x 5 minutes tranfsre with a 5-min pause in between.
Cheers.
Anna Konovalova can you please share what should be the final pH of this buffer?
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