amethyst73 | If the tech ain't broke, don't fix it!

I've been spending an inordinate amount of time over the past few weeks throwing my ~120 IgSF proteins into Drosophila S2 cells, and running a whole lot of Western blots to figure out which ones are expressed and which ones aren't.

In the lab that I work in, just about every protein we make has a 6-Histidine tag at the very end. It's exactly what it sounds like - a string of 6 histidine residues - and is useful for purifying your protein over a nickel column, or being able to detect all your proteins with an antibody to 6-His. You can throw any protein with a 6-His tag onto a Western blot, stain it with an anti-6-His antibody, do a secondary stain with an HRP-conjugated antibody, toss a compound on top that glows when it's hit with HRP, and expose the whole thing to film. The film will have dark bands wherever your protein is on the blot, and the intensity of the band will - very roughly - indicate how much protein there is. The proteins all need that tag, though, and you won't be able to tell anything about what other tags your protein is carrying.

To keep the explanation that follows relatively simple, I'm going to group my constructs into two classes, A and B. The A constructs have one particular set of 'tags' on the end - things like the 6-His tag I just described, which are helpful in purification and detection and such. The B constructs have a different set of tags from A. Every single one of my constructs has a 6-His tag on the end, no matter what other goodies it's got. When all is said and done with my 120 IgSF proteins, they all look like:

IgSF protein - group A - 6-His

and

IgSF protein - group B - 6-His

This is cool and useful in a number of ways, most of which I won't go into here. But having these two different tag sets means that I can toss proteins that have group A or group B onto the same gel, stain the Western with two different antibodies (each specific to a particular tag in either group A or group B), do a secondary stain with two additional antibodies (each of which sticks specifically to one of the first two). Each of these secondary antibodies fluoresces at a particular infrared wavelength, and can be detected with the neat new two-channel infrared scanner that the department just purchased from LiCor. Group A-tagged proteins show up as green bands, group B as red. This potentially becomes very useful if you want to detect levels of both group A and group B constructs in the same sample.
So largely because the fancy scanner is a new toy, I've been double-staining all my Westerns and visualizing them that way. Using this technology led us to the conclusion that only around 65% of our constructs are expressing. That's... lower than we want for our big upcoming experiment. A lot lower. Group B in particular appears to have low expression using this method.

(For the record, it should be noted that our antibody for Group B is kind of dirty. It leaves a high background, which means that bands are hard to visibly detect against the background. It's possible that if we found a better antibody for Group B, this method would work better.)

Just for the heck of it, we decided to try running a bunch of our Group B proteins that didn't appear to express using the infrared technology, using the old 6-His antibody with chemiluminescent technology. As you might have guessed from the title of the post, we saw expression of virtually everything we ran. This is pleasant - it means that our actual percentage of expressing proteins is rather higher than we originally thought. (It's also mildly aggravating in that we're going to have to rerun a lot of our stuff. It's not two weeks wasted, but it's always a little irritating to find out that a sizeable chunk of time could have been better spent.)

So.... with the antibodies we had on hand, looks like Old Tech For The Win. It should be noted, however, that Westerns are by no means the only thing you can throw on the IR scanner. You can scan stained gels (nucleic acid or protein), reporter assays, and mice. That's right - mice. Isaac Roger Photon, the official spokesmouse for LiCor's biotech product line, has his own pages at LiCor and on FaceBook.
But now I am at home, on another required vacation day, waiting for the tree guys to finish their work so I can go do stuff. My right arm is pleasantly sore from having played FFT A2 for too long. Opened up the Fusilier jobline!