Re:why I can see my band dyed in proceau S on PDFC?

proteins-request,

I have met a similar problem. Membrane I used was 0.45um pore size PVDF from Pierce. After transfer, the prestained ladder could be seen, but after a brief rinse, most of the prestained ladder was gone (Transfer buffer Towbins buffer with 10% methanol and 0.05% SDS). Also I have never seen such
things with NC membrane. I am wondering if it is caused by the SDS present in the buffer. However, in the instruction of PVDF membrane from biorad, methanol and SDS would not interfere binding of proteins to PVDF membrane. Any comments or suggestions?

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Sincerely,¡¡¡¡¡¡¡¡¡¡

Lu Falong
Group 808
Institute of Genetics and Developmental Biology, CAS
Beijing, PR China
[Only registered users see links. ]
2006-11-26

In article <[Only registered users see links. ].net >, "Lu Falong" <[Only registered users see links. ].cn> wrote:

It is caused by high pH. Ponceau S only binds to proteins at acidic
pH. At pH > 7, it's affinity for protein is too low and the stained blot
destains. You need to wash the stained blot with distilled water only.

DK




However, in the instruction of PVDF membrane from biorad, methanol

Lu Falong wrote:


SDS is used to mobilise large proteins from the gel, methanol to
increase binding of small ones to the membrane. Since they have opposite
effects there is little point in having both present at the same time,
even though a lot of people do that.

Blotting works better with Dunn's buffer (10 mM NaHCO3 and 3 mM Na2CO3,
Anal. Biochem. 157 (1986) 144-153) than with Towbins (and the former is
much cheaper, too).

If you have problems with Ponceau staining try india ink but beware that
that is a permanent stain that interferes with immune detection. Also
some of the fluorescent dyes marketed by Molecular Probes
([Only registered users see links. ]) are very sensitive.

In general you can expect better blotting results from PVDF than NC
membranes, as both affinity and capacity are higher. But note that not
all PVDF membranes were created equal, from the limited range I tried I
got best results with Immobilon P (Millipore). YMMV.