How to use separation glue?
Western blot membrane transfer
Under the action of current, the protein is transferred from the gel to the solid carrier (membrane).
Membrane selection: the commonly used solid-phase materials in imprinting include NC membrane, DBM, DDT, nylon membrane, PVDF, etc. We choose PVDF (polyvinylidene fluoride), which has better protein adsorption, physical strength and better chemical compatibility. There are two specifications: immobilon-p (0.45um) and immobilon PSQ (0.2um for MW < 20kDa).
A semi dry method
That is, the gel interlayer combination is placed between the filter paper with the transfer buffer, and the current is conducted through the buffer adsorbed on the filter paper to achieve the transfer effect. Because the current acts directly on the film glue, the transfer conditions are relatively harsh, but the transfer time is short and the efficiency is high.
(1) selection of experimental conditions
The current is 1ma-2ma / cm2, and we usually use 100mA / membrane. The transfer time can be selected according to the size of the target protein molecule and the gel concentration, and can be adjusted appropriately according to the actual situation.
Target protein molecular size (kDa), gel concentration and transfer time
80---140 8% 1.5-2.0
25---80 10% 1.5
15—40 12% 0.75
<20 15% 0.5
(2) experimental operation
(1) Preparation of filter paper and membrane (preparation shall be started 20 minutes before electrophoresis).
A. Check whether there are enough transfer buffers, which are not prepared immediately.
B. Check whether there is filter paper and membrane of proper size.
C. Immerse the membrane in methanol for about 1-2 minutes. And then transferred to the transfer buffer.
D. Soak the appropriate adhesive filter paper and membrane filter paper into the transfer buffer respectively.
How long should the PDVF membrane soak in methanol when entering the transfer buffer?
PVDF is hydrophobic, and it is difficult to soak in the transfer buffer. After methanol treatment, it is easier to soak. The pretreatment of PVDF is to soak and activate it with methanol, and then wash it twice with distilled water after soaking thoroughly. The purpose of using methanol bubbles is to activate the positively charged groups on the PVDF membrane and make it easier to bind with negatively charged proteins.
People usually use it for 5-20 minutes. It can be soaked at the same time as taking the glue, and it is estimated that it will take about 5 minutes. The effect is not bad. In the past, some webfriends posted that the processing time didn't matter much, but the key was the quality of the film. That's true. As long as the membrane is completely soaked, it should be OK.
Hybond's PVDF specification reads as follows: "pre wet the membrane in 100% metanol (10 seconds)". My understanding is that it's OK to soak for at least 10 seconds. In fact, it's OK to soak for 10 seconds or 10 minutes.
(2) Transfer
A. Lay the lower filter paper on the electric transfer instrument. Generally three layers are used.
B. Lay the membrane on the filter paper against the membrane, pay attention not to have bubbles between the membrane and the filter paper, and pour some transfer buffer onto the membrane to keep the membrane wet.
C. Peel off the glue, remove the stack gel and carefully move it onto the film.
D. Cut off the upper left corner of the film and mark the glue position on the film with a pencil.
E. Cover a piece of filter paper against the glue on the glue. Pour some transfer buffer and lay two pieces of filter paper.
F. Install the electric transfer instrument and select the required current and time according to the needs.
G. During the transfer process, the voltage change shall be observed at any time, and any abnormality shall be adjusted in time.
Post time: Sep-07-2022
