Loading Buffer, Laemli stain

Hello all,
I have tried many recipes that can be found through the net on how to
make a Laemli buffer for transferring proteins onto nitrocellulose but
most of them failed to give me the wanted result.
Do u have any valid recipe of making it?

Thanx

Ippokratis Messaritakis
Microbiologist, PhD Student
University of Crete, Faculty of Medicine
Dept of Bacteriology, Parasitology, Zoonoses and Geographical Medicine
Vasilika Vouton
71100 Heraklion, Crete
Greece
Tel. +30 2810 394572
Fax. +30 2810 394740

"Ippokratis Messaritakis" <[Only registered users see links. ].uoc.gr> wrote in message
news:[Only registered users see links. ].n et...

what problem are you finding?

Jose

Ippokratis Messaritakis wrote:


Does your problem occur during loading, staining or blotting? You seem
to be mixing up a couple of things here.

There are prestained molecular weight markers available, which allow you
to monitor correct separation during electrophoresis.

Once that works satisfactory, you have to stain your gel to verify that
you have bands in your sample. Since you want to do a Western afterwards
you need a non-denaturing stain, (copper, zink-imidazole or - slightly
more sensitive but much more expensive - one of the Sypro fluorescent
stains offered by Molecular probes).

If that worked without misshap, you can blot your gel, there are
essentially two procedures for this: that described by Towbin et al.
(Laemli running buffer without SDS, methanol may be added for better
transfer of small proteins) and that described by Dunn (NaHCO3/Na2CO3).
Of those the latter is cheaper, usually gives better results and is less
well known. Transfer can be either in a tank system or semi-dry, I get
much better results with the tank.

Proper transfer is verified by staining the membrane with Ponceau red.
The gel may be stained with Coomassie, if there are bands left, transfer
time needs to be increased. If the gel is clear but no bands are on the
membrane, you have most likely assembled the sandwich the wrong way
round, with the gel, rather than the membrane facing the positive
electrode.

If that hurdle has been taken too you can do the immunostaining for
specific proteins, at least in your positive control (you did include
both positive and negative controls?) you should see the band in
question.

If you go about this in a logical, step-by-step manner, your instructor
and we will be able to offer specific advice in case of problems.