Interpretting co-IP & other pulldown figures

Co-IP & other pulldown figures can get complex, but the key things are to:
1. Know what’s serving as bait - what’s getting directly pulled down? This should be labeled on the top right
2. Look at what’s being probed in the bound stuff you pulled out along with the bait (the “IP” columns). This should be labeled on the left-hand side. Each row will have a different one.
3. Look for differences in the input & IP
1. Loss of a band - no interaction
2. Maintenance of band - interaction
3. Relative strength of band in input vs. IP - strength of interaction
4. When you spot a difference, look to the top to see what changed in terms of included components.
full post: blog: bit.ly/pulldowns TH-cam: • Antibody biochemistry ...   • Pull-down assays (co-I...  
The more of the antibody-targeted thing there is, the stronger the band you see. If a protein was bound by the bait, you will see a band for it in your pull-down lane (the stuff you eluted) but if it wasn’t*, you won’t.   
  
*if you don’t see a band in your pull-down it could also be for a more boring reason - that it wasn’t even present in the sample (maybe those cells don’t express it). This is one reason why it’s so important to take and check that input sample!   
  
The strength of a band in the input tells you about how highly expressed a protein was. But the more exciting info comes from comparing that to the strength of that band in the pull-down lane. For the bait (bead-targeted protein), this just tells you about recovery efficacy: how well it bound the beads For other proteins ("prey"), this tells you about how strong the interaction with the bait protein was.  
  
And of course you also need to check your negative control lane. You don’t want to see bands here! If you see bands there, you probably need to wash your beads better and/or pre-clear your lysate with decoy beads, because stuff is sticking non-specifically to the beads.   
  
Before getting too excited about an interaction you think you’ve discovered, be sure to validate it. A common way you can do this is by swapping which is the bait and which is the prey. If you capture what was the prey does it pull down what was the bait with it? (E.g if A pulls down B does B also pull down A?) If so, it’s more likely the interaction is legit. At least in the lysate or whatever. Another hurdle to show is whether the interaction happens naturally in cells. Proving this might include things like in-cell immunofluorescence and/or various proximity-dependent readouts.   
  
more on western blots: bit.ly/westernblotworkflow ; TH-cam: • Western blots: the wha...   
  
more on loading controls: bit.ly/housekeepinggenes ; TH-cam: • Housekeeping genes/pro... & bit.ly/western_reprobe ; TH-cam: • Probing a western blot...   
     
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@MrBernarrr]

Hey Brianna,
I'm a die-hard synthetic chemist, but I've recently dived into the world of biomolecules. So, I'm still getting a grip on various biochemistry terminologies. Your video provided a great opportunity to improve my understanding using practical examples-thanks for that! Before watching your content, I turned to ChatGPT-4 with the image processing tool to clarify the Western blotting image for Flag-p65 from a streptavidin-biotin pulldown assay. I found ChatGPT's explanation refreshingly simple. However, since I'm still new to this, I'm uncertain how accurately it interprets the assay results. I'd love to see your take on ChatGPT's ability to analyze Western blotting with image processing. Keep up the good work!

[@thebumblingbiochemist]

Thanks! I've never used ChatGPT sorry!

[@ayavec]

Your videos help a lot ,
Thanks a bundle .
May I ask you to talk about the basics of flow cytometry , channels, and analysis, please 🙂

[@thebumblingbiochemist]

Glad to hear it! I don't know very much about flow cytometry sorry

[@kharaf9920]

thank you