![]() ![]() I dyed my membranes on Ponceau after transfer. I thought that my samples could be low on proteins, but why is the protein standard also not moving to the membrane? My samples got around 1 - 2 micrograms of protein per microliter, and I load each well on the gel with 5 - 10 microliters of sample. I thought about using wet transfer, but I read that this one is more suitable for High MW proteins, which is not the case for Gal-3, while semi-dry is more suitable for low MW. I could use this as a tiny little victory.Ĭan you guys share any thoughts on what I may be getting wrong, please? I truly have no idea what to do now. Clean as they can be, but the gel was no longer yellowish on the sides. In the end, again, both the gel and the nitrocellulose membrane were just as good as new. Then, I tried again, but only with Gal-3, and this time I used 2.5 A constant, up to 25 V and 7 minutes. After that, no protein was found anywhere, and my gel got a little yellowish on the sides, so I thought that the time for transfer was too much. The first time I transfered my samples, I've used 25 V constant, up to 1.0 A and through 15 minutes. ![]() Gal-3's MW is 31 kDa and TLR4's MW is around 90~100 kDa. I have always used the roller to remove bubbles on every step of the assembly. My transfer stacks and nitrocellulose sheet all were submerged in transfer buffer for at least 5 minutes prior to transfer. I assembled my blot sandwich according to instructions: on the bottom, a stack of transfer pads (which comprises of 7 layers of filter pads), followed by my nitrocellulose sheet, followed by my gel, followed by another transfer stack. trans-blot turbo mini size nitrocellulose trans-blot turbo mini size transfer stacks Bio rad 5x transfer buffer (when I dilute it, I put ethanol, following their instructions, and water) 4~20% SDS-PAGE Gels (I also used one made by myself. I'm using Bio-Rad equipment and reagents, being them: My gel gets cleaner as it can be, and my membrane gets nothing. I get to see all the bands from my molecular weight standard on the gel, everything is just fine, but when I put it to transfer on a semi-dry apparatus, they just fucking vanish. The different membrane options include nitrocellulose, PVDF, and low fluorescence PVDF.I need to do a western blot (fuck this assay) of two proteins (namely Gal-3 and TLR-4), and I'm having an awful ride when transfering them.ĭuring electrophoresis, everything runs smoothly. Whereas the Trans-Blot Turbo transfer packs come as pre-wet and assembled sandwiches, the new RTA transfer kits require membrane sandwich assembly by the user. They are offered in packages of 40 blots, consisting of membranes, filter paper, and a specially formulated transfer buffer. The new Trans-Blot Turbo RTA transfer kits offer the same performance at a more affordable price. Compared to traditional tank blotting, which typically takes 60 minutes to several hours, the Trans-Blot Turbo system and the associated transfer packs cut blot transfer time to as little as 3 minutes without sacrificing transfer quality. Bio-Rad’s latest semi-dry transfer system, the Trans-Blot Turbo, has changed the way researchers think about western blot transfers. ![]()
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