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Commercial western-blot membranes

Commercial western-blot membranes - Western Blot Forum

Commercial western-blot membranes - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 02-15-2012, 09:59 AM
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Question Commercial western-blot membranes



Hi!

Because of the difficult to obtain human tissues, in my lab, we are thinking to use commercial western-blot membranes.

I have found several types of them in different companies as Millipore, Imgenex or Gbiosciences.

Anyone have use some of these membranes previously? Which one are the best for WB experiments?

Thanks in advance!

Best,

Maria
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Old 02-15-2012, 09:41 PM
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Default Re: Commercial western-blot membranes

PVDF (Immobilon-P) from Millipore works well.
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Old 02-16-2012, 08:18 AM
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Default Re: Commercial western-blot membranes

I think that I've been misunderstood. The membranes, which I'm talking about, have an homogenate from different human tissues transfered in them. I'll attach a link to one of them. I'd like to know if someone have previously used some of these membranes and what kind (or brand) works better.

[Only registered users see links. ]

Thanks.
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Old 02-16-2012, 08:21 AM
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Default Re: Commercial western-blot membranes

I think that I've been misunderstood. The membranes, which I'm talking about, have an homogenate from different human tissues transfered in them. I'll attach a link to one of them. I'd like to know if someone have previously used it and what kind works better.

[Only registered users see links. ]

Thanks.
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  #5  
Old 02-16-2012, 10:08 AM
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Default Re: Commercial western-blot membranes

Protocols for Western Transfer with PVDF (Immobilon-P) from Millipore firm and nitrocellulose from other firms are the same with a few exceptions. PVDF is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes. Another change to note is that the SDS tolerances are not equivalent for the two membranes. The binding of protein to Immobilon-P is much more sensitive to SDS levels. Too much SDS can inhibit the protein's ability to bind to the PVDF and can, in fact, help proteins already bound to the membrane to slip off. SDS levels should never exceed 0.05%.
1) Optional: soak the gel in semi dry transfer buffer (48 mM Tris, 39 mM glycine, 0.037 % SDS, 20 % methanol) for 10-20 minutes.
2) Cut the membrane (Immobilon-P/PVDF) and 6 pieces of blotting paper (e.g. Schleicher and Schuell GB002) to the same size as the gel (~8.5 x 5.7 cm). Wet the membrane for 15 seconds in 100% methanol, then soak for 2 minutes in water, then equilibrate the membrane for 5 minutes in transfer buffer.
3) Assemble the transfer stack:
a) Three pieces of blot paper soaked in transfer buffer
b) Gel
c) Pre-soaked membrane
d) Three pieces of blot paper soaked in transfer buffer
4) Place stack upside down (gel side up) on blotter.
5) Blot at 0.8 mA/cm2 (~38 mA per gel) for 1-2 hrs.
6) Place the membrane on a piece of blot paper and dry for 2 hrs (or longer) at room temp or soak the membrane in 100% methanol for 10 sec. and dry 15 min.

good job Maria
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