Western Blotting Using the SemiPhor Semi-Dry Transfer Unit
Assembly of the Transfer Stack
- Remove the stacking gel from the gel. Measure the gel and record its size. Do not pre-soak the gel. Do not cut a corner off the gel, as this may allow a short circuit and inefficient transfer. Blot the gel as soon as possible after electrophoresis to avoid diffusion of the samples through the gel.
- Cut pieces of blotter paper to the size of the gel. You will need four pieces, plus one for each gel transferred. It is important that the blotter paper (and the membrane) not be larger than the gel. Larger pieces may make contact around the gel, and allow the current an alternate route, thereby making transfer inefficient.
- Wearing gloves rinsed of powder, cut a piece of nylon membrane to the size of the gel. Mark the lower right-hand corner to allow for orientation. Pre-wet the membrane in transfer buffer for 2 to 5 minutes.
- Place a mylar mask in the bottom of the SemiPhor unit. The mask should have a central, square opening cut in it that is 2 mm smaller than the gel in both length and height.
- In a dish of transfer buffer, saturate two pieces of blotter paper cut in step 2. Place these on top of the mylar mask, centering them by first placing the center of the paper down first, and rolling the edges out. The filter paper should cover the cut-out in the mask and slightly overlap it on all sides.
- Construct the first transfer sandwich on top of the previously-placed blotter paper by placing the membrane, then the gel, then an additional piece of buffer-soaked blotter paper on the stack. Up to five gels of the same size may be transferred simultaneously. If this is the case, place a sheet of cellophane (cut slightly smaller than the gel, it will swell when wet) on top of the first transfer sandwich, and construct the next one on top (membrane, gel, blotter paper; see diagram).
- Stack the components neatly, with edges parallel. As each layer is placed upon the stack, make sure no air bubbles are trapped. Sweep a wetted, gloved finger over the layer, or roll a pipette or test tube over it to remove bubbles. Proteins on the surface of the gel will bind to the membrane as soon as contact is made. The membrane must therefore be positioned correctly the first time. Do not try to adjust.
- Place two additional pieces of buffer-soaked blotter paper on top of the stack. Place the cover on the unit (it fits only one way). Hold the cover level and slide it gently down onto the stack.
- Do not remove the cover until after the blot is completed. Part of the stack may stick to the cover or otherwise be knocked out of alignment.
- When transferring multiple gels, it may be desirable to weigh down the lid in order to ensure even contact with the stack. In this case, place a weight (up to 1 kg) on the cover. Too much weight will compress the stack and hinder transfer.
- Connect the unit to a suitable power supply. Turn the power supply to zero before turning on. Turn on the power supply and set to approximately 0.8 mA/cm2 of gel. 8 mm x 7 mm mini-gels may be run at 100 mA constant current. Larger gels must be limited to 0.8 mA/cm2 to avoid excessive heat build up. Allow the transfer to proceed for the length of time specified in the table.
Approximate duration of transfer runs according to molecular weight: a
Molecular Weight Transfer Period <20,000 15 minutes 20,000-80,0000 30 minutes >80,000 45 minutes a 8 mm x 7 mm mini-gels at 100 mA.
Transfer Buffer (500 ml, pH 8.3) glycine 1.450 g (39 mM) Tris base 2.900 g (48 mM) SDS 0.185 g (0.037%) methanol 100.00 ml (20%) Combine reagents in a final volume of 500 ml dH2O. Use electrophoresis grade SDS. The methanol is not necessary when using positively-charged membranes.
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Revised: Sunday, September 03, 1995 1:24:52 PM