My molecular techniques is rusty. Been years since I've read Gene Cloning and DNA Analysis by TA Brown; might pick up the latest version.
A bait/prey setup comes to mind, but would be difficult to do for many proteins... hope there are better approaches
Thx!
In many papers, the author something along the line with this sentence "bound protein was subsequently eluted using an imidazole gradient (10–200 mM)". Am I right that they just make some buffers with increasing concentrations of imidazole, for example, 20 mM, 50mM, 100 mM, 150 mM and 200 mM? or do they use an instrument that allow them to elute the bound proteins with buffers with many more imidazole concentrations (10mM, 20mM, 30mM, etc)?
Also, is getting the target protein with a very low concentration a concern if you use so many buffers like that? In my experience, I always lose quite a lot of the target protein in the wash fractions.
Hello everyone! I’m relatively outside of my field doing some protein purification so please bear with me. I have a 2 kDa peptide that contains an N-terminal GST and His tag so it behaves as a protein in its original form. I am trying to purify and cleave the GST. We’ve seemed to optimize cleavage according to a gel I ran but I’m having trouble getting an efficient purification with a pure final product.
Originally I did a His tag purification with some help from an experienced person (so have complete confidence in the protocol). It seemed like we had product based on the FPLC chromatogram and gel but unfortunately nothing seemed to show up when running down my HPLC, either not enough sample was recovered or there were some impurities but very weird.
Which begs the question, for a small “protein” like this is it more efficient to purify by GST or His tag? I only have a His trap at the moment but can potentially purchase a column for GST capture. If I do so and have both options available for use, which order would be preferable (GST then His trap or vice versa). Any input would be greatly appreciated!
Hello! I am working on a research project where I'm determining which proteins are secreted by my microbe of interest during infection. To do this, we are adding different tags to the protein via translational fusions to pick up secretion. I would like to run my putative proteins through a protein modeling software with and without the different tags to see if the tags may be causing conformational changes to the proteins of interest.
I've tried Phyre and Swiss-Model, but they both either show me a predicted structure for my protein of interest, or the tag. I need it to show me a predicted structure for the entire peptide (aa sequence of putative protein + aa sequence of the tag).
Does anyone have any suggestions??
I'm studying fluorescent labeling of SNAP-tag fusion proteins for live cell imaging. Can you please explain why when the covalent bond is formed, the molecule will fluoresce?
https://preview.redd.it/nnwreeb30v661.png?width=637&format=png&auto=webp&s=fbaabc8ff116b6861674bea20d0d968bcd2afb72
pMAL vector has N-terminal MBP but I need 6HIS-MBP. I figured the way around it is to try using the vectors MCS to insert 6HIS tag at the c-terminal of MBP (MBP-6HIS) followed by my protein of interest.
Has anyone tried doing this successfully? Is there a stretch of intervening sequence that's required for the 6HIS to not be hidden?
I have never done any protein expression before and I've literally been thrown in at the deep end!
I'm trying to express an antibody in BL21 DE3 from a pET22b vector. My antibody should have an N terminal MBP tag and a C terminal His tag. I ran my uninduced and induced whole cell extract on a western blot today. I am getting a faint band for my uninduced and induced samples at ~42 kDa which suggests it's just MBP without my antibody attached. However, I'm getting a single band with anti-His at the right kind of size for my antibody (~17 kDa).
I have 5 vectors each expressing a different antibody and they are all doing it. The anti-His bands are slightly different sizes which is a bit concerning.
How am I getting His-tagged antibody protein without the MBP?
Get on YouTube and learn how to make a really nice omelet and/or make poached eggs. These are so good and versatile, and people can be just as impressed as if you made them a souffle.
Potatoes are also very cheap, and easy to learn to make in a nice fancy way, check out recipes for Patatas Bravas. But really you can just chunk them up and roast them in the even with olive oil and kosher salt and people will rave.
If you can splurge a bit on something, get a really good loaf of fresh fancy bread from a local bakery, or just a fresh (made that day) baguette.
Serve people an omelet with fresh herbs, oven roasted potatoes with a bit of spice added, a few chunks of fresh bagette, and a bowl of hulled sliced strawberries and they will feel like they are getting a big treat. All for potentially less money than even a hambuger and hotdog cookout.
A bargain bonus is to ask others to bring the booze for the eye-openers, people will likely ask what they can bring and this is the easiest thing.
Journal of the American Chemical SocietyDOI: 10.1021/jacs.9b12002
https://ift.tt/39vqcek
I've created some FKBP/GFP-tagged lines where the protein I'm tagging is smaller than the tag I'm adding. I can image them well but I'm wondering how would this affect the function of the protein? Some studies have that even just GFP alone can interfere with protein function but I haven't seen any that discusses a case where the protein of interest is smaller than the tag. Thanks!
