Transfer buffer in western blotting1/11/2024 Shake/rock the membrane in this for 30-45 minutes at room temp. (7) After a few minutes, pour off the first blocking solution and cover the membrane with fresh blocking buffer, this step helps to reduce the amount of the free antigen in solution. Even a small bulge from a plastic mold can push the membrane out of the solution and you will get a huge black mess on your blot when you develop it. During these steps, do not let the membrane dry. Transfer the membrane to a plastic tip box and cover with 10-15 mLs of blocking buffer. This step greatly reduces the smearing of the bands and the "flaming" appearance because it pre-coats adjacent binding surfaces. Before removing the membrane from the gel, pour some blocking buffer onto the blot. It's easier to peel the membrane off the gel. (6) Disassemble the stack by reversing the assembly order. Using milk is common and usually fine, but it can impede detection of some epitopes such as His 6-tags. Each is inexpensive and we have had the best performance with fish gelatin (it also smells like seagull lunch). Lately, we almost always use either spray-dried egg white powder, or fish gelatin. We used to use 0.2-0.3 grams of BSA or lysozyme in 50 mLs of TBST (expensive). (5) While the proteins are transferring, prepare a blocking buffer. Connect the power cables and transfer at a constant current of 1-2 mA per cm 2 for about an hour. The manufacturer mentions in the manual that the screws are rarely needed. You don't need the screws, in fact they will squeeze the buffer out of the gel and cause short circuiting. Also, this ensures a wet contact with the stack. The jerks in our lab rarely rinse off their buffers from the electrodes so things accumulate on the plates. Using the same Kim-wipe, dip it in some leftover transfer buffer and wipe/wet the upper electrode in the lid where it is going to contact the gel. I place a large crumpled Kim-wipe against the edge of the stack and roll toward it, this wicks away excess buffer than would otherwise squeeze out and short-circuit the transfer when the heavy lid is placed on top. Use a smooth cylinder (test tube usually) rinsed in a tray of leftover transfer buffer to squeeze out any bubbles. If you have multiple stacks to make, prepare them sequentially so the membranes don't stay exposed to the air. Immediately place two more pieces of paper on the membrane. Try to get it right the first time, proteins on the surface of the gel will immediately stick to the membrane and cause ghost bands in the Western if it's moved. Use a razor blade and pinch off the excess gel so its dimensions match the paper's dimensions. Carefully place the gel on the papers in the same way. The idea is to prevent bubbles from getting trapped. Hold the paper by the edges and "bow" it so the center touches first, then release it. Place two sheets (consecutively) on the bottom electrode. (3) Pour transfer buffer on the papers you cut and bring everything to the semi-dry transfer box. (2)ĝisassemble the gel, cut off the stacking layer, and soak the gel in about 30-50 mLs of transfer solution on a shaker for more than 5 minutes (In my hands, cutting this step causes blotchy Westerns and irregular transfers, free SDS in the gel will compete for protein binding). Don't wet the papers yet.ġ mM EDTA (I add EDTA, some people don’t, I find is seems to keep the 10X from turning yellow when stored for long periods)ġX transfer buffer is 1X Towbin and 20% methanol. Place the membrane in buffer on a shaker. Soak the membrane in 100% methanol for a few seconds, then pour off the methanol (I reuse mine) and immediately cover the membrane with 1X transfer buffer (below). Also, cut a piece of PVDF membrane that is slightly larger than the paper you just cut. While your gel is running, prepare 4 sheets of 3MM whatman paper with slightly smaller dimensions than the resolving layer of your gel. See Sauer:bis-Tris SDS-PAGE, the very best for my favorite SDS-PAGE technology. (1) Resolve your proteins by whatever gel technology you prefer. We use affordable in-house chemiluminescent reagents described in another protocol here. There are many commercially available technologies for generating a signal after the antibody is bound to the target protein, but our lab primarily uses chemiluminescent reagents and antibodies covalently conjugated to horseradish peroxidase. “Western blotting” is a common name used to describe a technique of detecting specific protein epitopes using antibodies.
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