Use separation glue
utions and common problems:
1) The size between filter paper, glue and membrane is generally filter paper > = membrane > = glue.
2) There must be no bubbles between filter paper, glue and membrane, which will cause short circuit.
3) Because of the hydrophobicity of the membrane, the membrane must first be completely wetted in methanol. In addition, the film must be kept wet at all times in the subsequent operation (except for the dry film method).
4) The filter paper can be reused. There are many transferred proteins adsorbed in the upper filter paper (membrane), so the upper and lower filter papers must not be mixed. If they cannot be distinguished, the rubber filter paper can be replaced.
5) The transfer time is generally 1.5 hours, (1ma-2ma / cm2, 10% gel), and the transfer time and current can be adjusted according to the molecular weight.
1) When laminating film, glue and paper sandwiches, operate in a large plate with a rotating film buffer. After stacking, transfer the three to the rotating film device in parallel. Do not move them any more, because the possibility of bubbles has been completely eliminated by operating in the buffer, so there is no need to drive bubbles
2) About film transfer time: 20 V for semi dry rotation × 30min
(b) wet method
We basically do not use this method, so we will not introduce it here.
C identification of effect after metastasis
1. Glue dyeing
After staining with Coomassie brilliant blue and decolorizing with Destain, see whether there is still protein on the glue to reflect the effect of transfer.
2. Dye film
There are two kinds of dye solutions, reversible and irreversible. Reversible ones include ponceau-s red, fastgreen FC, CPTs, etc. after dyeing with these dyes, the pigment can be washed away, and the membrane can be used for further analysis. But irreversible dyes, such as Coomassie brilliant blue, India ink, amido Black 10B, etc., and the stained film cannot be used for further analysis.
Post time: Sep-09-2022
