Electrotransfer experiments on proteins

Summary

Many membranes can be used as solid-phase supports for protein electrotransfer, such as diazotized cellulose membranes (DPT, DBM), DEAE-cellulose membranes, nylon membranes, etc., but the most used one is the nitrocellulose membrane (NC membrane), which binds to proteins in the form of noncovalent hydrophobic interactions with a binding capacity of about 80 μg/cm2.

Operation method

basic program

Materials and Instruments

Protein
Transfer Buffer Methanol Electrode Buffer
Electrotransfer Tank Electrophoresis Apparatus

Move

1. Cut six pieces of 3 mm filter paper and one piece of NC film.
2. Soak the cut 3 MM filter paper and NC membrane in transfer buffer for 3-5 minutes.
3. Install the transfer device as follows, placing the plastic holder flat in the tray containing the transfer buffer and placing a sponge on top of the plastic holder.
4. Place three pieces of 3MM filter paper on the sponge in pairs, and then place the NC membrane, gel, and the other three pieces of paper and sponge in turn.
5. Clamp each of the above layers with a plastic holder and place them in the electrotransfer tank, with the NC film side toward the positive pole and the gel side toward the negative pole.
6. Turn on supply voltage 120 mA and transfer for more than 6 hours or overnight.
7. After the transfer, take out the plastic holder, remove each layer in turn, mark the upper edge of the membrane with a pencil, cut off half of the membrane corresponding to one of the holes, and check the effect of the transfer by staining it with aminoblack or Coomassie Brilliant Orchid.
8. Place the remaining NC membrane on a clean 3 MM filter paper and dry at room temperature for 30-60 minutes (optional).

Caveat

1. The size of the paper and membrane should not be larger than the size of the adhesive to be transferred, otherwise a short circuit will occur during the transfer process, affecting the transfer effect.

2. Wear gloves during the operation, do not touch directly with your hands, because the sweat and secretion on your hands will directly affect the transfer of protein from the gel to the membrane.

3. When placing the NC membrane and filter paper, there should be no air bubbles between each layer, otherwise the transfer effect will be affected.

4. The molecular weight of the protein to be transferred determines the transfer time; a higher molecular weight results in a longer transfer time, and vice versa.

5. Although drying can make the protein more firmly bound to the membrane, it may also aggravate the texture denaturation of the protein and affect its immunoreactive activity.

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