Ponceau staining - what, why, & how with some practical tips

Рет қаралды 4,634

Жыл бұрын

A Ponceau stain is a quick, easy, and reversible way to check your membrane after protein transfer in a western blot to get a sense of how things went. It helps you see if there are bubbles, whether you loaded even amounts, whether the lanes rain evenly, etc. It’s a total protein stain so it will show you all that’s there. It isn’t very sensitive but it does the trick!
blog: bit.ly/ponceau_practical
basic protocol:
- run SDS-PAGE
- blot (transfer to membrane)
- rinse off in ddH2O (that really pure water) → shake ~1 min
- pour on enough Ponceau solution to coat the membrane → shake a couple min
- pour off Ponceau (can reuse lots of times)
- rinse background off with ddH2O
- take a picture
- wash all the way out with TBST
- block
- do your antibody probing stuff
more on western blotting: bit.ly/westernblotworkflow ; KZfaq: • Western blots: the wha... & more practical lab tips and tricks here: bit.ly/lab_tricks_page

more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

Пікірлер: 15

@sunnyx3511 Жыл бұрын

Really helpful for my practical exams ❤️

@caucasianeagle5320 Жыл бұрын

Hi, Just did a western blot today and with my collegue we didn't know what to do after staining with ponceau. Washed couple times with water and worried if we lost some proteins but as soon as we put it into tbst it got clear. I wish I could have watch this video before. I would like to thank you. You're my hero. I don't know how many methods and tricks I learned from you. If I know why I am doing something most of the time because of you.

@thebumblingbiochemist Жыл бұрын

I'm soooo happy to hear you find my content helpful. Best of luck with your research!

@TALKyLIKEENGLISH Жыл бұрын

Very helpful. I wanted to ask why i had unspecific bindings of antibodies to many proteins at one even though i did blocking fir an hour. What can be then the probklem?

@thebumblingbiochemist Жыл бұрын

you might not be washing enough

@Howling-wolf. 3 ай бұрын

thanks

@Aash0999 Жыл бұрын

Thank you for this informative and helpful video! Could you please share your tips for separating large proteins? What are your recommendations in terms of time, voltage ..etc, for the running and transfer steps? I've been struggling with separating a large protein (>200kDa). Thank you always

@thebumblingbiochemist Жыл бұрын

Glad you found it helpful! I don't have experience with large proteins like that but if you do some Googling you can probably find some tips. Sorry I can't help more!

@NewWesternFront Жыл бұрын

I only do a few rinses with dH2O on the Ponceau, leaving the membrane stained. To the effect that the blocking solution has a pink tint everytime i've poured it off. Seems to have worked fine so far. What do you think?

@thebumblingbiochemist Жыл бұрын

Could probably potentially obstruct binding a bit but not a huge deal

@chelodiaz7288 Жыл бұрын

hey, have been watching your videos for a long time, and i am having trouble with a western blot, i charged a pre stained protein ladder, (which is visibly transfered) but when i do the western blot i can't see the proteins after the reveal, it's weird because i can see the proteins on the gel after the transference if i apply coomasie blue. Any tips? Thanks a lot and your videos , they are very helpful.

@thebumblingbiochemist Жыл бұрын

Hi. Are you saying your ladder is transferred but not the rest of your protein? So the ladder doesn't show in your gel but the proteins do? All sizes? Did you try an unstained ladder as well? Did you do any sort of fixation that could have affected things?

@TALKyLIKEENGLISH Жыл бұрын

Cool❤