Pull-Down Assay Protocol

Protein interactions reveal a lot about how proteins and cells function under different conditions. One tool that allows us to look at direct protein interactions is called a pull-down assay. A pull down assay utilizes a bait protein bound to beads in a column to catch protein binding partners. This technique can be used to verify a predicted protein interaction via Western blot or identify novel protein interactions using a total protein stain.
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[@youtubeidfor8076]

amazing, I'm a first-year researcher but I've never done pull-down assay, so i've been always confused about the concept of Pull-down, and bait and prey proteins,,, and it always made reading articles difficult, but you make me perfectly understand it!! Thanks a lot.

[@kharaf9920]

I do not generally comment but you explained it brilliantly where many others failed. you deserve much more subs/views !

[@CCheiley]

I totally agree with you!

[@SENSRF]

Thank you for your feedback! we hope you check out more of our content on our channel!

[@pranaygaikwad2129]

Extremely lucid explanation about pull down assay.. Thanks for this.. Hope that you will continue making videos on many other techniques.

[@gariyoshi95]

The best one ever I have seen so far !!

[@thecerebrum.]

Thanks for making this!

[@shumailakhanum2279]

You made it quite simple n easy to understand

[@juangalindo6398]

Beautiful!🤜🤛🙌

[@lesquisse8898]

Thanks a lot!

[@kuerove]

Amazing!

[@shrishtiwari7750]

Excellent

[@ahmadmuhammad.0163]

amazing

[@MasterWaterBender]

Thanks ❤

[@DeaDCellbio]

that was very useful Thanks

[@andrasbajusz9290]

Lets say we found that there is the same proteins in the control and the experiment collum. How do we know that those protein can not connect to the bait?

[@SENSRF]

Thank you for your question! Ideally, you wouldn’t find the same protein in the control and experimental column because in the control, the bait isn’t adhered to the column, and therefore can’t catch the prey. Any proteins that do appear in the control are non-specifically bound, meaning that when analyzing proteins bound to the experimental column have to be disregarded. If your protein of interest non-specifically binds to the control column, you would need to troubleshoot to assess if the column is suitable for your protein (there are a few different kinds, some of which are stickier than others).

[@andrasbajusz9290]

@@SENSRF I see, thanks!

[@queenchicken6430]

Very well explained! What kind of staining do you use to reveal unknow binding proteins ?

[@SENSRF]

Thank you for your question. Most of the time, when scientists do a pull-down, they run them on a gel and then stain it with an antibody against predicted binding partners - much like a western blot. However, when scientists are conducting an exploratory assay to find unpredicted (but previously discovered) binding partners from the column, they will often run all proteins eluted off the column in a size-selection gel, stain it with silver stain, and then select bands of interest to run through mass spectrometry, which quantifies the proteins mass to charge ratio and then compares that to a database of known proteins. For uncharacterized/unknown proteins, there are some novel protein identification methods (including protein sequencing) using mass spectrometry. Let us know if you have any further questions!