Pour off the TBST buffer.Īdd Blocking Buffer (BSA in TBST) and block for 1 hour at RT.ĭilute Primary antibody in the blocking buffer according to the manufacturer’s ratio. Pour off the Ponceau S staining buffer, wash 3 x 5 minutes with TBST buffer. Pour off the water and submerge the blot in Ponceau S staining buffer within the cleaned plastic tray, incubate 5-10 minutes on a rocker (or manually shake back and forth). Optional: Or 100 V for 1-2 hours, depending on protein size.ĭay 2: Blot Incubation with Primary antibody probesĭisassemble the sandwich in a tray of dH2O and remove everything except for the blot. If you don’t have a cold room, you can place the tank in an esky filled with cold packs with the wires leading out. If you have a cold room, move everything there now. The side of the sandwich with the nitrocellulose membrane should be closest to the red! Run towards red! Place the cassette and the ice block into the tank together, then put on the lid. Lift up your transfer sandwich by squeezing the fiber pads on either side, then move the entire sandwich to the cartridge/cassette that will hold it for electrophoretic transfer. The final product should look something like this (top to bottom - ie. Soak a second filter paper square, smoothing it over the membrane, then push the second thick fiber pad into position. Soak the transfer membrane next, then move it into position above the gel and smooth it down. Next move your SDS-PAGE Gel across under the water until it is lying square on top of the filter paper. Next, gently push the first piece of whatman paper down onto the fiber pad, smoothing out any air bubbles. Start by placing the first fiber pad into the tray of transfer buffer, pushing it right to the bottom. Trying to manually move it with your hands will nearly always end in disaster.Īssemble your transfer sandwich, carefully ensuring there are no air bubbles between any layers. You can photograph your gel with white light before continuing but you risk tearing it.įill the plastic trays with 1 x Transfer buffer to a depth of 1-2 cm, then gently push the SDS-PAGE gel from its cassette into the tray using water.Īn excellent technique for moving an SDS-PAGE gel without tearing it is to use a wash bottle to push it along with a jet of water. Load the protein extracts on an SDS-PAGE gel, run at 100V for ~1 hour. Perform a cell lysis protocol to lyse your cells. Protocol: DAy 1: Cell Lysis, SDS-PAGE, Membrane Transfer Ice Block (this part will need to be frozen beforehand) One of these two will likely have the positive and negative electrode Remove the membrane with forceps and rinse it in deionized water.3% Bovine Serum Albumin added to TBST bufferĪdditional Primary/Secondary antibody pairs if you wish to search for additional proteins/include a control.Ĭhemiluminescent substrate appropriate for Secondary antibodyĪppropriate illumination source for the substrateĢ x large plastic/glass tray large enough to hold 4 SDS-PAGE gels side by side. When the transfer is complete, turn off the power and peel off the layers of the sandwich until you reach the membrane. Run the transfer unit at 320mA for 1 hour.
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Press to engage the latches with the guide posts without disturbing the gel/nitrocellulose stack. Soak another piece of blot paper and place on top of the gel, carefully removing air bubbles from between the gel and filter paper.Ĭarefully place the cathode plate onto the stack. Roll a pipet or test tube over the surface of the paper (like a rolling pin) to exclude all air bubbles.Ĭarefully place the equilibrated gel on top of the nitrocellulose membrane, aligning the stack as perfect as possible. Place this pre-soaked sheet of blot paper onto the platinum anode. Prepare in advance the nitrocellulose and filter/blot paper.Īfter running an SDS/PAGE gel, immediately equilibrate the gel in a small container of Semi-dry transfer buffer for ~15min.Ĭompletely saturate a piece of blot paper by soaking in transfer buffer.
![western blot sandwich western blot sandwich](https://www.leinco.com/wp-content/uploads/2020/04/WesternBlotSetup-02-scaled.jpg)
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