May 27, 2026

Recombinant Protein Purification Master Protocol

This  protocol  is a draft, published without a DOI.
  • Ainsley Lederer1,
  • Prem Shrestha1,
  • Ji Youn Park1,
  • In-Kwon Kim1
  • 1University of Pittsburgh
Ainsley Lederer
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Protocol Citation: Ainsley Lederer, Prem Shrestha, Ji Youn Park, In-Kwon Kim 2026. Recombinant Protein Purification Master Protocol. protocols.io https://dx.doi.org/
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 21, 2025
Last Modified: May 27, 2026
Protocol  Integer ID: 233194
Keywords: recombinant protein purification, recombinant protein purification master protocol, recombinant protein purification master protocol this protocol, freezing pure protein, maximum purity of resultant protein, denaturation of protein, different methods of purification, purification, pure protein, guanidine, chromatography, resultant protein, protein, maximum purity
Abstract
This protocol details the process of large scale recombinant protein purification from E. coli culture. This takes you from start (beginning the seed culture) to finish (freezing pure protein). We detail three different methods of purification (affinity, ion-exchange, size exclusion) to ensure maximum purity of resultant protein. This method does NOT involve denaturation of protein with guanidine-HCl.

The expected result? A pure protein that is active and stable! All proteins are different, and this is simply meant as a framework to take you through all the steps. Each step, especially in the chromatography, should be optimized to suit your protein.
Guidelines
You'll see me say this countless times throughout the protocol, but this protocol should ONLY be used as a framework/starting point, and should absolutely be customized to your protein and lab systems. Every protein is different, especially regarding items like IPTG concentration, induction temperature, buffer components/pH, etc..

It is essential to customize the protocol to your proteins' needs in order to achieve good yield and activity!
Materials
For E. coli culture:
  • 100 mL and 1 L flasks of autoclaved LB broth
  • 1000X antibiotics (100 mg/mL ampicillin, 50 mg/mL kanamycin, 34 mg/mL chloramphenicol, etc.)


NI-NTA BUFFERS:

Ni-binding buffer: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM imidazole, 10% glycerol, 5 mM beta-mercaptoethanol (BME)

Ni-washing buffer: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 20 mM imidazole, 10% glycerol, 5 mM BME

Ni-elution buffer: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 250 mM imidazole, 10% glycerol, 5 mM BME

ION-EXCHANGE BUFFERS:

Cation low salt: 25 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol, 5 mM BME
Cation high salt: 25 mM HEPES pH 7.5, 1 M NaCl, 5% glycerol, 5 mM BME

Anion low salt: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5% glycerol, 5 mM BME
Anion high salt: 50 mM Tris-HCl pH 7.5, 1 M NaCl, 5% glycerol, 5 mM BME

NOTE ON IEC BUFFERS: The principle of IEC relies on your electrostatic interaction between the charge on your protein and the charge of the column. Different amino acids have different pKas, contributing to your protein's overall pI. In order to have your protein be charged (i.e., able to bind the column), your buffer pH must be either 0.5-1 units higher (for anion exchange) or 0.5-1 units lower (for cation exchange) than your protein's pI. For example - if my protein has a pI of 6, I would want to use a heparin column and a buffer with pH 7.0.

SIZE EXCLUSION BUFFER: 25 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 2 mM DTT


Before start
Before you start, review all steps of the protocol and make sure you plan your timing accordingly. This whole process can take 3-5 days. All materials should be stored in the fridge throughout the entire procedure.

Lysing the cells open is the "point of no return" in this protocol, meaning after this step it is important to work as efficiently as possible to purify the protein so as to not risk degradation over time.
E. coli seed culture - Day 0
16h
Take your glycerol stock or colony from plate and inoculate 100 mL of LB media with the proper antibiotics. If using glycerol stock, don't thaw stock - just take a pipette tip, "scoop/scrape" some icy cells, and put the tip in the flask.
E. coli culture and induction - Day 1
Prepare your flasks containing 1 liter LB media by adding the proper antibiotics and any cofactors if necessary. In our lab, we generally use 4-6 flasks (so 4-6L total), depending on protein and desired yield.
Add 10-12 mL of your overnight culture to each flask.
Note
For many incubators, flasks that are near the heating element will have faster growth. so we add 10 mL to these flasks and 12 mL to the flasks further away to equalize growth.

Transfer flasks to a floor shaker. Set temperature to 37 °C and shake flasks at 200 rpm for ~3 hours.

Take a measurement of the OD600 after ~3 hours. The target OD600 for most proteins is 0.6-0.8.
If your OD600 is less than this, you can calculate the approximate time the cells will reach the target. For example, if your OD600 is 0.15, it will take 2 doubling times to reach 0.6. If your E. coli are doubling every 20 minutes (this is the typical doubling time), then you should measure the OD600 again in 40 minutes.
Once the OD600 reaches 0.6-0.8, lower the temperature to18 °C and induce the cells with IPTG and any other necessary additives. Allow cells to incubate with IPTG for 16-18 hours.

If using BL21 BirA cells, add biotin to 50 micromolar (µM) final at this step too. Typical IPTG concentration is 0.5 millimolar (mM) final, but this can vary based on your protein. Do a literature search or test expression beforehand to optimize. Induction temperature also may vary. Again, optimize beforehand!



Cell harvesting and purification - Day 2 to finish
The day after inducing, remove flasks from incubator and prepare for centrifugation. The method you follow will differ based on your rotor, culture amount, and available bottles/tubes. For a TX-1000 rotor, divide the culture into four 750 mL bottles. Weigh on the balance to ensure even weight. Centrifuge the bottles at 3500 rpm, 4°C, 00:45:00 .

In a sink with running hot water, pour the supernatant out of the bottles, leaving a layer of cells on the bottom behind.
Transfer bottles to ice. Add 25 mL of Ni-binding buffer to one bottle. Vortex the bottle at speed 7 until cells are thoroughly resuspended. Transfer this suspension to the next bottle. Repeat and continue transferring suspended cells until all bottles have been resuspended.
Divide your cell suspension into two 50 mL conical tubes. Centrifuge tubes at 6000 rpm, 4°C, 00:10:00 . Discard media completely by turning tubes upside down on a paper towel. Be careful! If the pellet is soft, don't let it slide onto the paper towel.
Note
NOTE: At this point, pellets can be frozen at -20ºC and stored for several days before proceeding with next step.


Place the conical tubes on ice and add 35 mL Ni-binding buffer to each tube. Add benzamidine to 1 mM final and Aprotinin to 7 ug/uL final. If you are working with a sensitive protein, you may want to add additional protease inhibitors.
Resuspend the pellets by vortexing at speed 7.
After resuspending, lyse the cells with sonication. The sonicator tip size and settings will depend on the system you use. For QSonica Sonicator, we use the largest (1/4 inch, 4435) probe. The sonication settings are: 7:00 minutes, 10s on 40s off, 70% amplitude.
Transfer lysed cells to ultracentrifuge tubes. Reserve a few uL of lysate to run on gel later if desired. Spin the ultracentrifuge tubes at 18000 rpm for 30 minutes. We use a Type 50.2 rotor with 39.2 mL QuickSeal tubes.

Ni-NTA Purification
Collect soluble fraction (supernatant from ultracentrifuge step) in a tube or bottle. Using a washed/equilibrated 5 mL Ni-NTA column and peristaltic pump, flow the lysate through the column and collect in another bottle or tube. Use a flow volume of ~2 mL/minute or less.
Note
Note - there are many ways to use Nickel affinity resin. Of course, this assumes you have a histidine-tagged protein. The easiest method is to use a packed column and peristaltic pump. If unavailable, use "loose" resin with batch purification or gravity method. See our other protocols for more information.

Additionally, be careful - large proteins (>80 kDa) typically don't tolerate the pressures of a peristaltic pump, and require batch or gravity Ni-NTA purification.

Once the lysate has flown through and been collected, flow through 35 mL Ni-binding buffer and collect.
Flow through 35 mL Ni-washing buffer and collect.
Elute your protein with Ni-elution buffer in three separate tubes/steps: 5 mL, 15 mL, and 10 mL. The majority of your protein should be in the second tube.
Run a gel to confirm protein presence and successful isolation. Typically I run the "total lysate" (collected before ultracentrifuge step), "soluble lysate" (supernatant after ultracentrifugation), collected flow through from loading the lysate onto the column, binding buffer sample, washing buffer sample, then the three elution samples.


Expected result


Above is just one example of a gel result from Ni-NTA chromatography. In this case, I did not run the "total lysate" (sample that would've been saved in step 14 of cell harvesting section). As you can see, the majority of my protein is in the second elution fraction.

You should not expect 100% purity after Ni-NTA. This chromatographic step is done as a first-pass purification to remove most, but not all, proteins and contaminants.



Ion Exchange Chromatography
Either look in the literature or use ProtParam to estimate your protein's pI. Based off the pI, choose whether you will use anion-exchange or cation-exchange. The two columns we use in our labs are "Q" and heparin, respectively. From here, you should use the buffer that corresponds to the type of ion-exchange you are doing. See more information on buffer pH in the materials section.
If your protein is present in the Ni-NTA elution samples, proceed with IEC chromatography. Combine the fractions from Ni-NTA which contain your protein, and add IEC low salt buffer for a total volume of 40-45 mL*.
Note
*This is a general guideline. If your protein binds weakly to IEC column, you may want to supplement the elution fractions with a "No salt" buffer rather than low-salt. The purpose of this step is to dilute/equilibrate the protein sample in the buffer which will be used for IEC, to ensure correct pH and charge of the protein.

Flow the protein sample through the IEC column using a peristaltic pump and collect the flow-through. After, flow through an additional 10 mL of low salt buffer.
Use an FPLC system to run ion-exchange by eluting with a gradient of high salt buffer.

Depending on your chromatography system, the protocol which you use will vary. Refer to your lab manual for specifics. If you do not have an FPLC, you can elute with a step-wise gradient of salt concentrations.

Specific to our lab: we use a Bio-Rad NGC system. To run IEC, you first need to exchange the buffers on top of the FPLC for your low and high salt buffer, and pull air out using the syringe with luer lock. Then, purge the system. After doing this for both pumps, you must wash the tubing using the prescribed settings in our protocol. Once washed, you will attach the column, set up fraction collection, and run the FPLC. NEVER use the FPLC without training. If you are unsure of the settings/protocol, ask a senior lab member for help.

After viewing the results of the IEC chromatogram, run a gel on any fractions which may contain your protein. I also recommend to run a sample of the flow-through from loading the IEC column. Some proteins may not bind to the column, and that is okay. IEC chromatography is still recommended to remove impurities like other proteins and nucleic acids.
Expected result

This is one example of an ion-exchange chromatogram. The blue line is the UV/vis A280 measurement of any proteins which are eluted from the column. The red line represents the conductivity, i.e. the salt concentration. This protein is a weak binder, as it eluted very early in the salt gradient application.


Size Exclusion Chromatography to finish
After identifying which fractions contain your protein from IEC, as well as purity, concentrate the protein using a centricome. The MWCO will depend on your protein size. Refer to the manual for the specific centricome you use for details on spin speed and sample type. In our lab, we use 2000-3000xG in a swing bucket rotor.
Note
Centrifugal filters can be reused several times if stored correctly. Always wash the filter with buffer after concentrating a protein, and store the filter with MilliQ water in the fridge. To check filter integrity, you can use BSA or similar and run a gel on the filtrate.

Continue to concentrate the sample until you reach the required volume for your FPLC loop and column. For protein yield more than 1 mg, we use a superdex 200 HiLoad 16/600 column with a 5 mL loop. For 1 mg or less, we use Superdex 75 increase GL 10/300 with a 1 mL loop.
Split the concentrated protein solution into 1.7 mL microcentrifuge tubes, and spin down at 14000 rpm for 10 minutes at 4C. This ensures any sediment or aggregates will not be injected into the FPLC.
Using a washed injection needle and syringe, aspirate the protein samples, leaving behind any aggregates on the bottom of the tube. "Flick" the side of the syringe to dislodge any bubbles and let them go to the top, towards the needle. Once bubbles are at top, gently push on syringe and observe bubbles coming out of needle. Slowly continue to push until only liquid, and no bubbles are coming out. This step is crucial for ensuring bubbles are not injected into the FPLC.
Once the FPLC is set up (buffer has been exchanged, proper lines are connected to columns), inject the sample and start the run.
After the run has completed, run a gel based on the chromatogram. Protein should be pure. If it is, concentrate the protein again using centricomes and measure the protein concentration (using Nanodrop, Bradford assay, etc.). Aliquot the protein to your desired volumes and flash freeze with liquid nitrogen, before storing at -80ºC.
Expected result

Example chromatogram of size-exclusion results. For the column used (Superdex 200 pg HiLoad 16/200) there is a loading step, and a collection step. The loading step flows buffer through the column for 120 mL (i.e. the column volume). After 120 mL of buffer, collection starts for 180 mL.


Give yourself congratulations! You are a protein purification master!