Dima Klenchin wrote:
>>tdschr0 at pop.uky.edu ("Todd Schraw") wrote:
> :Having similar problems with transfer efficiency of a 200 kDa protien.
> :Referencing "Molecular Cloning, A laboratory Manuel " (Sambrook, Fritsch,
> :Maniatis) They suggest an effective range of separation of
> :SDS-Polyacrylamide Gels as follows:
> :
> :15% = 12-43
> :10% = 16-68
> :7.5% = 36-94
> :5% = 57-212
>> These numbers are significantly off IMHO.
>
completely off, right. With the conventional Tris-Glycine buffer, a 10%
SDS gel will be sufficient for most purposes with proteins in the range
of 35-150kD. For proteins smaller than 35kD, a 12% SDS-gel is good. For
proteins <20kD it is better to use a "Schaegger-style" tris-tricine
SDS-gel. For resolution of proteins >120kD, a 7% Gel is good, or - for
proteins >250kD - a polyacrylamide/agarose composite gel.
> :They also recommend the following Transfer Buffer:
> :39 mM Glycine
> :48 mM Tris Base
> :0.037% SDS (electrophoresis grade)
> :20% Methanol
> :
> :They have the transfer running with a current of 0.65 mA/sq. cm. of gel
>> That would be per sq.cm of gel's cross-section (width x thickness),
> not just "gel".
>
Hmmm, not sure. We always use 0.8-1mA per sq.cm of gel (width x height),
and I wouldn´t really understand what the thickness of the gel would
have to do with it. Of course, a thicker gel takes somewhat longer to blot.
> :Another aspect that they repeatidly point to is the possiblity of a short by
> :using nitrocelluse or stack paper that is larger than the gel itself. The
> :make a big deal about having these exaclty the same size as the gel.
>> This is simply so wrong, it's not even funny. I never woudl have
> thought to find this in Maniatis.
>
right.
It may be different for semidry blotting, when too thin stacks of paper
are being used, or the lid is improperly closed, allowing for a direct
contact between the electrodes. If that was not the problem, too big
papers would simply increase the area and call for a higher current.
> For 220K protein I would try any combination of the following
> (in the order of preference; if first does not help, combine it
> with the next): 6% gel, strongly alkaline buffer (like CAPS pH
> ~10), twice longer than normal transfer time (make sure cooling
> is OK), 0.01% SDS in transfer buffer.
CAPS buffer is a really good chaoice for blotting, but the time needed
for a complete transfer is way shorter than with the conventional
buffer. We use a 10mM CAPS, 10%MetOH, pH 11 buffer and blot for 15
minutes. The drawback of this buffer is that you have to prepare it
fresh each time, as CAPS is not stable at RT. So you cannot have a
convenient stock, or a ready-to-use bottle of transfer buffer. That´s
why we only use it when the protocol calls for it (e.g. for Edman
sequencing the protein, where glycine should of course be avoided...)
Frank
