Protein purification Puns

I’m going to be purifying a human ABC membrane transporter protein as part of my PhD work. My lab/PI hasn’t done this before, anybody have any links to resources to start figuring out the basics of protein purification and expression and how to plan this? It’s a heavily studied protein with lots of protocols for expressing and purifying it already but I’m unsure how to select the best methods for my application

I'm doing protein purification using urea. I have done so far 5 times, with pretty much the same steps, except that the starting OD of IPTG induction and the lysing time may vary a bit. However, there was only one time that the target protein was eluted out in the 500mM imidazole and urea.

I am using an IMAC resin, and the only concentration that can elute only my target protein is 500mM. In all trials, about 80% of my target protein remained bound in the resin.

Do you always have this issue? or do you think it's my fault to not keep the whole experiment the same?

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.

Sorry, complete noob with protein purification…

I’ve been using an IgG HRP conjugation kit and their reducing agent seems to have become less reliable as of late (we ran a cross reactivity test with a previous lot’s reducing agent and the conjugation was much better) As a result, I’m losing about 30% of my IgG-HRP conjugates.

If I were to purify these by FPLC to make sure I get rid of any unconjugated IgG, would I just need to set up an FPLC column with anti-HRP IgG?

I'm searching for some practical manual or review on different buffers used for protein purifications. We have some know how in my lab of course but I would like to found something to read to get some different ideas on how to deal with my targets.

I've found some more theory focused book and some review on very specific application, but I would like something more practical, with some concrete example.

Do any of you guys have a source?

Does anyone know any good resources to learn some biochemistry related to protein purification? My project involves the purification of a functional membrane protein and I would need to purify several other soluble proteins later down the line. Although I've taken several biochem courses in the past, I still don't know the practical details (ie which component to add to the buffer) to consider during protein purification. My entire lab is not a biochem lab, so the help I can get from my PI is minimal. What are some good resources (videos, websites, webinars, or books) to learn more about the topic?

Any input is greatly appreciated!

Hey guys! I'm but a humble cell biologist trying to do protein purification for the first time, and it's proving a little frustrating. My PI also has little to no experience purifying proteins. Help?

I'm trying to purify 3 GST-tagged proteins (same protein family) from bacterial lysates using glutathione agarose 4B resin in spin columns. The proteins express great and survive pelleting, but during purification it looks to me like they are degrading significantly. On Coomassie gels, there are low intensity correct bands, along with massive bands at 25 kDa which I can only assume must be GST only? My proteins are also about 24 kDa so would be in the same respective bands. I don't add any protease (there is a PreScission site) as I actually need to keep the GST tag for my assay. There are also a couple of other lower bands which I assume are degradation products.

Here is my protocol - does anyone know what could be happening?

1. Wash 1 mL glutathione sepharose 4B resin in PBS twice and keep on ice
2. Add 20 mL lysis buffer (300 mM NaCl in 50 mM sodium phosphate pH 8 with 2 mM PMSF) to the frozen pellet and gently scrape+pipette to resuspend on ice
3. Sonicate resuspended pellet at 25% amplitude, 2 minutes, 2 seconds on/2 seconds off
4. Centrifuge lysate at 12,000 x g for 30 minutes with the centrifuge set to 4 C
5. Collect the supernatant and gently resuspend the washed resin with it
6. Incubate the supernatant+resin on a rocker at 4 C overnight
7. Pour the supernatant+resin gently into a centrifuge column and spin at 500 x g for 1 minute at 4 C (collect some flow-through)
8. Wash with 5 mL room temperature PBS and spin at 500 x g for 1 minute, then repeat 4 more times
9. Elute the bound protein with 1 mL 10 mM glutathione (reduced) in 50 mM Tris-HCl (pH 8), then repeat 5 more times, all at 4 C
10. Add sodium azide (0.02%) and keep elutions in the fridge (short term)

It just confuses me that there is all this GST-only despite not being intentionally cleaved and the full-length protein expressing fine in the cells. What could be going on?

I'm designing protocol to purify protein fragments after degradation. My plan is to include a FH8 tag at the N-terminal of my construct and purify via affinity chromatography. My hopes is the fragments will break randomly and I'll be able to quantify where the sites of post-translational modification and/or degradation occured with mass spectrometry.

Example of mass spec results:

N-HHHHHHHH-Domain1-Domain2-Domain3

N-HHHHHHHH-Domain1-Domain2

N-HHHHHHHH-Domain1

Has anyone performed something like this? I appreciate your input! :)

Hi, I had a FT come service one of our Akta systems recently, and he was like “You’re have it easy for me! It’s not in a cold room.” So makes me wonder, what kinda proteins do you purify that needs a cold room? We do ours at RT. I know cold temp is better for stability and to slow down catalytic activity, but is there another reason to put a FPLC in there? I means it’s cold,...so cold.

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!

I am trying to catch my protein using a his-tag on a Ni2+ column. My protein is expressing great and is soluble. However when I go to put my sample through the column, most of the protein goes through in the flow-through but a small amount binds and can be recovered in the elution. What can cause not all protein to bind? I have tried extended time on the resin, so I know it’s not a time sensitive process. Additionally, I have used new resin, recharged old resin, and additional amounts of resin. Any ideas or suggestions would be appreciated.

I am currently using 50 mM phosphate, 500 mM NaCl, 8 M urea for lysis and refolding rapidly on the column with the same buffer (- urea). Washing with 20 mM imidazole and eluting with 250 mM imidazole. No protein is coming in the wash so there must be a problem with initial binding to the resin.

Update: higher pH in the lysis buffer and higher concentration of imidazole in elution did the trick. Thanks everyone!

Hey Lab rats,

Hope everyone is enjoying the last few days of this dreadful year (2020). I have a question to ask regarding my protein purification. My protein undergoes degradation while purifying during the Ni-NTA step. I have tried a bunch of protease (EDTA free) inhibitors cocktails but with no luck. I am still getting degraded bands right underneath my protein.

Note: My protein (66aa) has an N terminal His tag, followed by MBP (Maltose binding protein) and tev cleavage site. The protein is completely disordered (Hence the MBP tag).

Buffer used: 20mM TRIS pH 8.0, 500mM NaCl, 1mM TCEP, 10% Glycerol.

Let me know if you have any more questions!

Hello fellow labrats,

My colleagues and I have been largely successful at producing proteins and peptides using affinity tags. Our proteins are secreted by cho cells, not overly large, say 20 to 60 kda, and we use cell culture supernatant and run these gst fusion proteins on gst columns. We then get rid of the gst using on column cleavage by a protease. This strategy had worked consistently but obviously it contains the added constraints of getting rid of the gst. Any thoughts or strategies to produce native proteins without affinity tags and purify them. Using antibodies specific for proteins that are produced seems attractive but very expensive in a large scale set up. Any protein experts?

Hi all,

Due to Covid-19 our lab got strict occupancy numbers for all rooms but as we are a membrane protein lab you can imagine that our cold room was always very busy but now only one person at a time is allowed in there and most of us need to do their purification at said temperature.

We mainly do gravity flow affinity chromatography in there so I was wondering if you can do this partially at room temperature and just have the buffers on ice and elute into a falcon tube on ice and if this is enough to keep the resin cold. We could get cold cabinets but that would take a while.

Any short term solutions would be appreciated!

I work for a company that manufactures laboratory products (apparatus type of products, semi consumables racks, bottles, icewares, pipettes, flasks, beakers etc) I am hoping to chat with some people who have practical experience in cell culture and protein purification to see what products you are using and what you may like or dislike about them. Also hoping I am not breaking any sub-reddit rules with this post and if I am I do apologize. If anyone is interested please contact me. I would be very appreciative.

Hey guys, so this might come off as a bit of rant and asking for suggestions at the end.

First, the rant part. I have been training myself (with help from fellow labmates) on designing plasmid constructs for large-scale protein expression and purification in mammalian expression system (Expi293 suspension cell line). While the design part has been fine (cloning has been okay though could be laborious at times), when I got result from my transfection and checked the profile on SDS-PAGE, I got basically smear bands from the constructs I designed myself.

I felt a bit devastated because I spent a lot of money on the transfection. I am using Expi293 with manufacturer's recommended medium, the Expi293 growth medium, which is stupidly expensive. Not only that, the transfection reagent Expifectamine is also stupidly expensive. Felt like I just wasted about >$800 without getting usable product. As of note, I ran a 200 ml transfection volume. I got proteins secreted out of the cells and was able to purify with nickel resin (my protein is 6xHis-tagged).

So, I feel like next time I should do small scale first, but I do not know what would be the most optimal way of doing that. Here is my line of thought:

First, culture non-Expi293 cell line (maybe 293F FreeStyle) and use PEI or Lipofectamine or 293fectin. PEI is the cheapest among those 3, but some people complained about its efficiency. Maybe do it in 30 ml? Then, purify the protein in the supernatant, then run gel.

Does anyone have a better strategy that optimizes for speed and cost? Thank you!