List of questions:

How does one functionalize a protein?
How do you know what beads to use?
What is the significance to the different tags?
How do you know what beads to use? or columns? or anything else?
Does it matter what pool we use?
What is a good starting amount of pmol?
Do we need to start a new notebook, or can we use the old one?
Is the buffer they mention on the data sheet the one we should use?

*Reminder for Brad: Please send terrible binding assay results to Michael and I! :)


Mimi Le said...

I'm going to try to answer this as best as I can (meaning, this is what I would do in your case).

The type of beads used depends on how you functionalize a protein; so if you biotinylate your protein, you would use streptavidin beads; if you his-tag your protein, you would use nickel beads. Tagging your target protein allows you to immobilize the protein onto the correct type of magnetic beads. This way you can easily capture your target by using a magnet to separate what you want (which are attached to the magnetic beads) and what you don’t want. I don’t know if it matters what type of pool you use; I usually use what Brad has in stock (N44, N34, etc.) I’ve always started out with a 1:1 DNA/RNA to protein ratio, but you can change your conditions to test for the best ratio (though that may be tedious). If you have enough room in your old lab notebook to record information regarding the new selection, I wouldn’t bother getting a new lab notebook (especially if you’re using the same conditions/techniques that were previously pasted in the old notebook). If it specifies what kind of buffer the protein was made/suspended in, I would use that in your selection.

Nia_Fernandez said...

Before using exactly what Brad has in stock I would recommend talking to him or some of the other students about what has worked. cheesy enough "not all pools are created equal" and it has been shown that some of them don't work as effectively!

i agree with mimi 1:1 is usually your best bet! your first couple of PCR gels and definitely your binding assay will tell you whether changes should be made (ie very early amplification means tons of DNA/RNA is present)

Brad Hall said...

1.) To functionalize a protein, you typically use a biotinylated NHS ester that will bind to amines (R-N-H2 groups). Pierce makes the EZ-Link NHS-SS-Biotin. For this to work, you must have a target with an N-terminal amine (i.e every polypeptide) or a protein with an amino acid that contains amines (i.e. cysteine). The problem with functionalizing proteins this way is that you have to have the ratio of reagent to target just right to ensure you are attaching one or two biotins to the protein without wasting to much non-functionalized protein or biotinlyating the crap out of it.

2.) I will post a sign on the fridge about linkers/beads to use. In general if your target is tagged with biotin, GST, poly-his, or Fc complement you should be fine for our beads.

3.) Tags are typically used for purifying recombinant proteins. Some tags work better than others and the tag will determine the beads (see what mimi said).

4.) I defer to Mimi on the pool.

5.) The pmol to start should be ~400pmol, but you can get away with less or use a lot more. As Mimi said, 1:1 ratio is good, but not required. If is often useful to perform an initial binding assay to see if the target binds well to the pool or not. If the target binds well, you can get away with a lot less. If it doesn't bind well, you should use more target to encourage binding.

6.) Defer to mimi on the notebook answer, but make sure you tab separate your old and new work.

7.) The buffer can be useful, especially if it has some chemical the protein is stable in. But the general buffers we use in lab should be sufficient.