i'm not sure how hard you have to try, to press a high weight protein ALL the way through a membrane so i am rather inclined to believe that your current runs into the wrong direction.Įither your membrane/gel order is wrong or you plugged the cables into the wrong poles. also, i am going to go ahead and assume that you run the gels long enough for a 31 kDa protein not to run out.ĭo you have any controls that you stain for? what do you mean by 'nothing to be seen', literally nothing? if so than you either blot into the wrong direction or you blot too hard/too long. I assume that you blot them on two different membranes right? 31 kDa and 100 kDa is a bit far apart so be seperated sensibly on a single gel. Only thing I didn't to was Coomasie dye my gels after transfer, but for lord's sake, I can clearly see my MW standard ladder on the gel.Ĭould it be that I'm using too much filter pads?įor the records, I'm undergoing masters degree and my defense is just around the corner. 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. 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.
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