Name: an economical and versatile high-throughput protein purification system using a multi-column plate adapter
Uploaded: 2021-05-21T13:00:00.000+00:00
Duration: 10 min 8 s
Description: Watch this Scientific Journal Video about An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter at JoVE.com

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451 and an internal research grant from the University of Liverpool.

1. Denaturing Ni-NTA chromatography
<ol>
	<li>Preparing buffers 
	NOTE: See Table 1 for details of all buffers.
	<ol>
		<li>Make up 500 mL of 0.5 M NaOH, making sure to add the Milli-Q water first and then adding the NaOH slowly whilst the beaker is on a stirrer. Filter using a 0.22 &#181;m filter.</li>
		<li>Prepare 100 mL of 0.1 M nickel sulphate and filter using a 0.22 &#181;m filter.</li>
		<li>Prepare 50 mL of 2 M imidazole and filter using a 0.22 &#181;m filter.</li>
		<li>Prepare and 0.22 &#181;m filter 1.5 L of denaturing buffer at pH 8.0 consisting of 5.3 mM Tris-HCl, 4.7 mM Tris-base (Tris (hydroxymethyl)methylamine), 13.7 mM NaH2PO4, 86.3 mM Na2HPO4 and 6 M guanidine hydrochloride.</li>
		<li>Prepare and 0.22 &#181;m filter 500 mL of lysis buffer solution of 10 mM imidazole dissolved in denaturing buffer (see 1.1.4) using the 2 M imidazole stock.</li>
		<li>Prepare and 0.22 &#181;m filter 500 mL of wash buffer solution of 10 mM imidazole dissolved in denaturing buffer (see 1.1.4) using the 2 M imidazole stock.</li>
		<li>Prepare and 0.22 &#181;m filter 500 mL of 100 mM EDTA buffer, adjust pH to 8 using 1 M NaOH.</li>
		<li>Prepare and 0.22 &#181;m filter 500 mL of elution buffer made up of 7 mL of 99% glacial acetic acid into 6 M guanidine hydrochloride. 
		NOTE: If using native buffers, adapt according to Table 1.</li>
	</ol>
	</li>
	<li>Lysing cells (denaturing method)
	<ol>
		<li>Add 2 mL of lysis buffer to every 1 g of harvested E. coli cell pellets that contain the over-expressed His-tagged protein. Vortex for several minutes until cells are re-suspended. Leave the samples on a rocker for 30 minutes at 4 &#176;C.</li>
		<li>Centrifuge lysed pellets at 18,000 x g for 30 minutes. Keep 50 &#181;L aside in -20 &#176;C freezer to analyze later on an SDS-PAGE gel.</li>
		<li>Carefully pour off the supernatants making sure that the pellet is not disturbed. Lysates should be a clear, light yellow color. If lysates are cloudy, centrifuge sample again; consider sonication if available to degrade nucleic acid.</li>
		<li>Filter the supernatants through an empty column (without resin but with filter) and collect in an open collection tray. Then transfer into a tube for use in step 1.3. 
		NOTE: If using the native buffers, then after step 1.2.1, treat the samples to 3 cycles of freeze/thaw at -80 &#176;C or process by sonication or French Press to lyse cells.</li>
	</ol>
	</li>
	<li>Preparing vacuum purification setup and equilibrating columns 
	NOTE: See Figure 1 for a set up MCPA with 24 columns.
	<ol>
		<li>Determine how many columns are needed as well as the desired resin type and volume. As a rule of thumb for 100 mL of autoinduction culture (~2 g pellet) expressing His-tagged SH3 domains using T7 based plasmids, use 0.6 mL of Ni-NTA resin per column to yield about 3 mg of protein from about 6 mL of lysate.</li>
		<li>Insert the desired number of 12 mL columns (without resin but with filter) into a pre-assembled MCPA (long-drip plate with punctured sealing mat). The columns should fit snugly in the holes of the sealing mat that have been punctured.</li>
		<li>If less than 24 columns are used, close empty holes with an empty closed column.</li>
		<li>Take an open collection plate and put this plate into the base of the vacuum manifold and close with the top of the manifold.</li>
		<li>Place the assembled MCPA with columns onto top of the vacuum manifold.</li>
		<li>Attach tubing from a vacuum pump system to the inlet/nozzle at the bottom of the vacuum manifold.</li>
		<li>Ensure that the Ni-NTA beads are in a 50% solution with 20% ethanol. Before doing anything with the beads gently shake/mix the bead/ethanol solution to resuspend evenly. Then, using a 5 mL pipette, pipette 1.2 mL of the 50% solution into the columns. Whilst pipetting make sure the beads remain fully mixed.</li>
		<li>After pipetting into all 24 columns, turn on the vacuum pump and run the 20% ethanol through the column and into the collection plate below.</li>
		<li>Turn off the pump once all the 20% ethanol has run through all the columns (Ni-NTA resin can tolerate brief drying if the vacuum is still applied after buffer has passed through the column). Dispose of what is in the open collection plate into a waste box. 
		CAUTION: All waste products from the Ni-NTA beads are hazardous, when handling the beads or disposing of the waste, wear gloves, goggles and other PPE. Furthermore, dispose of all Nickel Sulphate waste in a separate container for individual disposal later. 
		&#8203;NOTE: If resin is new or recently regenerated, the rest of these steps can be ignored, move to the next section.</li>
		<li>Add 3 resin volumes (1.8 mL) of 100 mM EDTA buffer using a 20 mL syringe or a repeater syringe device with a 50 mL syringe. Then switch on the vacuum pump to let the EDTA buffer wash through and switch off the pump once it has washed through. 
		&#8203;NOTE: This wash should remove the nickel blue color but if this is not the case then repeat this step until all columns have lost their blue color. Turn off the pump and pour the contents of the open collection plate into the waste box.</li>
		<li>Add 3 resin volumes (1.8 mL) of 0.5 M NaOH buffer into each column before turning on the pump and running this through each column. Empty collection plate.</li>
		<li>Add 4 resin volumes (2.4 mL) of 100 mM nickel sulphate into each column. Turn on the vacuum pump and run this through the column once again.</li>
		<li>Add 10 resin volumes (~6000 &#181;L) of Milli-Q water and running this through the columns. Turn the pump off and pour off the contents of the collection plate into the waste box.</li>
		<li>Wash the columns twice with 4 resin volumes (2.4 mL) of 10 mM imidazole in wash buffer (denaturing or native) each time to remove any excess nickel and equilibrate. Empty collection plate. 
		&#8203;NOTE: If any columns are running significantly slower, detach column and check air can be pushed through the MCPA using a large syringe. If this is unblocked, use a different column with a fresh filter.</li>
		<li>If multiple different samples/proteins are going to be purified on the MCPA then replace the open collection plate with a 48 x (5 mL/well) collection plate. However, when only running the same protein samples on every column, it is fine to carry on using an open collection plate.</li>
	</ol>
	</li>
	<li>Loading, washing and eluting
	<ol>
		<li>With vacuum off, load lysates into columns (the amount of lysate will vary, typically from 1 to 12 mL or more and may be required to load in several batches). Use a thin plastic stirrer to gently mix the beads and the lysate in the column to maximize binding.</li>
		<li>After gently mixing each column for a few minutes, switch on the vacuum pump and run the lysates through the columns. If multiple protein samples are being run, use a 48 well collection plate and make sure to swap out the collection plate for another 48 well plate after 4 mL has run through as the wells in these plates can only hold 4 mL of solution. Continue running lysate until all the lysates has been run through a column. Freeze collection plates that contain the flowthrough. 
		NOTE: Columns may become &#34;blocked&#34; causing the lysate to flow through the column much slower than it should. This is easily spotted as the neighboring columns will be flowing at a normal rate. If this does occur, it is recommended that the lysate/resin mixture be transferred to a column containing a fresh filter.</li>
		<li>Replace the collection plate with an empty open collection plate and then add 9 resin volumes (5.4 mL) of 10 mM imidazole wash buffer to each column and turn on the vacuum and run the wash through. Repeat this 4-5 times. To avoid overflow, periodically empty waste plate.
		<ol>
			<li>Then replace the collection plate with a clean appropriate plate (open plate for just one protein and 96 well if there are multiple proteins, ensuring that A1 is in the top left corner).</li>
		</ol>
		</li>
		<li>Using a repeater syringe with a 12.5 mL syringe, pipette ~1 resin volume (0.75 mL) of denaturing elution buffer into each column.</li>
		<li>To avoid protein foaming, turn on the vacuum pump at the lowest setting. Gently lift the MCPA to check nothing is left to drip into the collection plate. 
		&#8203;NOTE: An alternative to eluting with the vacuum or gravity is pushing a 10 mL syringe plunger into the top of the column to gently push the elution buffer through the column. Do this for every column, being careful when removing the syringe plunger not to disturb the beads at the bottom of the column. Once the elution buffer has been pushed through, gently remove the syringe plunger from the last column it was used in and briefly turn on the vacuum pump to remove the last drops from the columns.
		<ol>
			<li>If a 96-well collection plate has been used, then check the collection plate to ensure that what is flowing from each column through the long drip plate is only flowing into one well and nothing is eluting into neighboring wells on the collection plate.</li>
		</ol>
		</li>
		<li>For the next elution repeat the above 2 steps and collect in a fresh multi-well (different samples) or open collection plate (same samples).</li>
		<li>Optional step, take a 50 &#181;L aliquot of each elution for purity and concentration analysis.</li>
		<li>Add 2 mL of 20% ethanol to each column and run this through using the vacuum pump. Then add another 2 mL of 20% ethanol to the columns and use a fresh thin plastic stirrer to mix up the 20% ethanol and the beads before transferring to a 50 mL tube for storage at 4 &#176;C. Clean the columns and check that water flows freely through each column and then clean everything and put away for later use. 
		&#8203;NOTE: Some columns will eventually have their filters blocked and will run too slowly, these columns should be replaced, or filters cleaned. As such, it is recommended to periodically test filters by removing resin and filling the column with water to see if it flows through quickly. Filters can be cleaned by leaving a closed column filled with denaturing elution buffer and shaking overnight.</li>
	</ol>
	</li>
</ol>
2. Ion Exchange - Single protein purification
<ol>
	<li>Preparing the buffers 
	NOTE: See Table 1 for details of all buffers
	<ol>
		<li>Prepare a 2 L low salt buffer: 10 mM Tris pH 8.1 (5 mM Tris Acid, 5 mM Tris Base). Filter solution using a 0.22 &#181;m filter.</li>
		<li>Prepare a 0.5 L high salt buffer: 10 mM Tris pH 8.1, 4 M NaCl. Measure 116.9 g of NaCl and make up to 0.5 L with the 10 mM Tris pH 8.1 from above. Filter solution using a 0.22 &#181;m filter.</li>
	</ol>
	</li>
	<li>Making salt concentration series: 0-1 M NaCl. 
	NOTE: The range of salt concentrations can be adjusted for every protein.
	<ol>
		<li>Setup 24 x 50 mL labeled centrifuge tubes in a rack</li>
		<li>Using the buffers made above, prepare 24 x 14 mL NaCl dilutions ranging from 0-1000 &#181;M. Start by adding the required volumes of 4 M NaCl 10 mM Tris. This could be done by increments of 25 mM or 50 mM depending on protein.</li>
		<li>Then add the required volumes of 10 mM Tris to each tube. Invert each tube several times to mix.</li>
	</ol>
	</li>
	<li>Preparing samples
	<ol>
		<li>Obtain the samples to be purified by Ion exchange. Optionally, take a 50 &#181;L aliquot for analysis later. 
		NOTE: Samples should always be kept cold; these may be lysates, or maybe post Ni-NTA. Samples from Ni-NTA in denaturing buffers require dialyzing or buffer exchange in 10 mM Tris buffer. Samples from Ni-NTA in native buffers could be diluted with 10 mM Tris buffer to reduce the salt concentration so that the protein can bind to IEX resin.</li>
		<li>Spin samples at 18,000 x g for 10 minutes.</li>
		<li>Carefully pour off the supernatant into a weigh boat. Avoid disturbing the pellet.</li>
		<li>Take up the supernatant carefully into a 20 mL syringe and attach a 0.22 &#181;m filter.</li>
		<li>Slowly eject the supernatant out through the filter into a fresh 50 mL tube.</li>
		<li>If dilution is required, dilute supernatant with 10 mM Tris buffer made above (In this experiment the supernatant was diluted 1 in 2). Store at 4 &#176;C in the meantime.</li>
	</ol>
	</li>
	<li>Preparing the vacuum purification setup and equilibration setup 
	NOTE: In this protocol, only one column is used for IEX purification of one sample. For multiple purifications, see next section.
	<ol>
		<li>Determine the desired resin type and volume. As a rule of thumb for up to 20 mg of partially pure His-tagged SH3 domains, use 0.4 mL of Q-sepharose fast flow resin per column (see step 2.4.8).</li>
		<li>Insert 24 open empty columns (empty without filter at bottom) into a pre-assembled MCPA. The columns should fit snugly in the holes of the sealing mat that have been punctured.</li>
		<li>Place a 10 mL syringe plunger into every empty column except the first position, where the purification will start.</li>
		<li>Push the bottom of an open column (with filter) through a 5 mL syringe plunger rubber gasket (that has been pierced) to form a rubber ring at the end of the column. This rubber ring will ensure a good seal when this column is inserted into each empty column above. For now, insert this column into the first empty column on the MCPA.</li>
		<li>Take an open collection plate and put this plate into the base of the vacuum manifold and close with the top of the manifold.</li>
		<li>Place the assembled MCPA (with columns) onto top of the vacuum manifold.</li>
		<li>Attach tubing from a vacuum pump system to the inlet/nozzle at the bottom of the vacuum.</li>
		<li>Cut a blue pipette tip ~ 2 cm from the bottom and use to take up 800 &#181;L of Q-sepharose beads (or any other ion exchange resin), ensuring that the 50/50 bead-ethanol solution is mixed to avoid layering. Pipette 800 &#181;L into the one open column (with filter) in the first position and once the beads settle to the bottom of the columns switch on the vacuum enough to run through the 20% ethanol.</li>
		<li>Wash the column containing sepharose beads with 2 x 2 mL of 10 mM Tris. Turn the vacuum off just before the Tris buffer runs through to prevent the resin drying out. Run off can be discarded. 
		NOTE: If resin has been used previously, regenerate by washing in 5 resin volumes of 4 M NaCl and then 5 resin volumes of 10 mM Tris before step 2.4.9.</li>
	</ol>
	</li>
	<li>Loading, washing and eluting
	<ol>
		<li>Transfer all sample to be purified (see section 3) to the Q sepharose column in the first position, use a small plastic loop to stir sample and beads for around ~2 minutes before switching on the vacuum pump. 
		&#8203;NOTE: In cases of excess supernatant (&#62;12 mL), beware of overfilling the column. Let the sample run through then add more, mixing before letting run through again.</li>
		<li>Store/Freeze the flowthrough for analysis later.</li>
		<li>Place another open collection plate into the vacuum manifold and then wash each sepharose column with 2 x 2 mL of 10 mM Tris and store.</li>
		<li>Insert a 96 well collection plate, making sure A1 is in the top left corner.</li>
		<li>To collect the first elution fraction, remove the syringe plunger from the next column along, move the Q sepharose column into this position and put the syringe plunger in the previous position. Then pipette 1 mL of the first salt concentration into the Q sepharose column. Turn on the pump and collect the elution. Turn off the pump just as the liquid lines near the beads to avoids drying out the beads.</li>
		<li>To collect the next elution fraction, remove the syringe plunger from the next column along, move the Q sepharose column into this position and put the syringe plunger in the previous position.</li>
		<li>Add 1 mL of the next salt concentration to the column and repeat the process until all concentrations have been used and 24 elutions have been collected in 24 separate wells. Check no liquid has entered any neighboring wells.</li>
		<li>Wash the column with 2 mL of 4 M NaCl 10 mM Tris. Let this run through.</li>
		<li>Wash with 2 mL of 20% ethanol. Let ethanol run until it is just above the beads and seal column for storage.</li>
		<li>Store 96 well collection plate and analyze by UV spectroscopy and SDS PAGE.</li>
	</ol>
	</li>
</ol>
3. Ion Exchange - Simultaneous purifications of 24 different proteins
NOTE: See steps 2.1-2.3 for details on making buffers, a series of salt concentrations and preparing samples
<ol>
	<li>Preparing the purification setup 
	NOTE: How the purification system is set up is very much dependent upon how many proteins being purified and how many salt conditions will be used. To purify 12 samples up to the maximum of 24 samples simultaneously, a collection plate for every salt concentration used to elute is required. For less than 12 samples, it is possible to move the chromatography columns a few positions within the same MCPA before changing collection plates. In this case, there is no need to place the columns in empty columns and they can be directly attached to the MCPA sealing mat. The protocol below is for 24 simultaneous ion exchange purifications.
	<ol>
		<li>Place the assembled MCPA directly attached to 24 empty columns with filters onto top of the vacuum manifold that contains an open collection plate and prepare and wash 24 ion exchange columns as in single purification method above. For this purification, it is convenient to use an Eppendorf repeater or syringe to rapidly pipette into columns.</li>
	</ol>
	</li>
	<li>Loading, washing and eluting
	<ol>
		<li>Place a 48-well collection plate into the base of the vacuum manifold before fitting the MCPA (with the washed purification columns attached). Ensure A1 is in the top left corner.</li>
		<li>Pipette each protein sample (up to 5 mL at a time) into their corresponding column. Stir each column using a thin plastic loop (one loop for every column to avoid contamination) for approximately 2 minutes. Then switch on the vacuum pump and let the supernatant flow through. 
		NOTE: In cases of excess supernatant, beware of overfilling the columns. Let the sample run through its column then add more, mixing before letting run through again.</li>
		<li>Store/Freeze the 48-well collection plate for later analysis as this contains the flowthrough for every protein.</li>
		<li>Place another 48-well collection plate into the vacuum manifold and then wash each sepharose column with 2 x 2 mL of 10 mM Tris.</li>
		<li>Insert the first 96-well collection plate into the manifold, ensuring that A1 is in the top left corner and then pipette 1 mL of the first salt concentration into each column and then turn on the vacuum pump to run this through the columns. Remove the collection plate, cover with parafilm and store in at 4 &#176;C for analysis.</li>
		<li>Repeat step for each successive salt concentration used to elute. Make sure that a new collection plate is used for every elution and that each collection plate is labelled and stored.</li>
		<li>Wash each column with 2 mL of 10 mM Tris, 4 M NaCl.</li>
		<li>Wash each column with 2 mL of 20% ethanol. Let ethanol run until it is just above the beads and seal columns for storage.</li>
		<li>Store 96 well collection plates and analyze by UV spectroscopy and SDS-PAGE.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Automated+Mid-Scale+Parallel+Protein+Purification+System+for+Antibody+Purification+and+Affinity+Chromatography.">Development of an Automated Mid-Scale Parallel Protein Purification System for Antibody Purification and Affinity Chromatography.</a> Protein Expression and Purification. , 128 (2016).</li><li>Renaud, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+in+Drug+Discovery:+Impact,+Challenges+and+Opportunities.+Nature+Reviews.">Biophysics in Drug Discovery: Impact, Challenges and Opportunities. Nature Reviews.</a> Drug Discovery. 15 (10), (2016).</li><li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+Protein+Purification+and+Quality+Assessment+for+Crystallization.">High-throughput Protein Purification and Quality Assessment for Crystallization.</a> Methods (San Diego, Calif). 55 (1), (2011).</li><li>Pan, W., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Metal+Affinity+NCTR+25+Tag+as+a+Better+Alternative+to+the+His-tag+for+the+Expression+of+Recombinant+Fused+Proteins.">A New Metal Affinity NCTR 25 Tag as a Better Alternative to the His-tag for the Expression of Recombinant Fused Proteins.</a> Protein Expression and Purification. , 164 (2019).</li><li>Locatelli-Hoops, S., Sheen, F. C., Zoubak, L., Gawrisch, K., Yeliseev, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+HaloTag+Technology+to+Expression+and+Purification+of+Cannabinoid+Receptor+CB2.">Application of HaloTag Technology to Expression and Purification of Cannabinoid Receptor CB2.</a> Protein Expression and Purification. 89 (1), 62-72 (2013).</li><li>Pina, A. S., Lowe, C. R., Roque, A. C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+Opportunities+in+the+Purification+of+Recombinant+Tagged+Proteins.">Challenges and Opportunities in the Purification of Recombinant Tagged Proteins.</a> Biotechnology Advances. 32 (2), (2014).</li><li>Gaberc-Porekar, V., Menart, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+of+Immobilized-Metal+Affinity+Chromatography.">Perspectives of Immobilized-Metal Affinity Chromatography.</a> Journal of Biochemical and Biophysical Methods. 49 (1-3), (2001).</li><li>Anderson, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Process+of+Structure-Based+Drug+Design.">The Process of Structure-Based Drug Design.</a> Chemistry &amp; Biology. 10 (9), (2003).</li><li>Cummin, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+High-Throughput+Purification+Method+for+Hit+Identification+in+Protein+Screening.">A Simple High-Throughput Purification Method for Hit Identification in Protein Screening.</a> Journal of Immunological Methods. 339 (1), (2008).</li><li>Dominguez, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multi-Column+Plate+Adapter+Provides+an+Economical+and+Versatile+High-Throughput+Protein+Purification+System.">A Multi-Column Plate Adapter Provides an Economical and Versatile High-Throughput Protein Purification System.</a> Protein Expression and Purification. , 152 (2018).</li><li>Bolanos-Garcia, V. M., Davies, O. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Analysis+and+Classification+of+Native+Proteins+From+E.+Coli+Commonly+Co-Purified+by+Immobilised+Metal+Affinity+Chromatography.">Structural Analysis and Classification of Native Proteins From E. Coli Commonly Co-Purified by Immobilised Metal Affinity Chromatography.</a> Biochimica et Biophysica Acta. 1760 (9), (2006).</li><li>Ayyar, B. V., Arora, S., Murphy, C., O'Kennedy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+Chromatography+for+Antibody+Purification.">Affinity Chromatography for Antibody Purification.</a> Methods in Molecular Biology (Clifton, N.J.). , (1129).</li><li>Jubb, H., Higueruelo, A. P., Winter, A., Blundell, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Biology+and+Drug+Discovery+for+Protein-Protein+Interactions.">Structural Biology and Drug Discovery for Protein-Protein Interactions.</a> Trends in Pharmacological Sciences. 33 (5), (2012).</li><li>Grabowski, M., Niedzialkowska, E., Zimmerman, M. D., Minor, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Structural+Genomics:+the+First+Quindecennial.">The Impact of Structural Genomics: the First Quindecennial.</a> Journal of Structural and Functional Genomics. 17 (1), 1-16 (2016).</li></ol>

The authors have nothing to disclose.

The method is robust and simple to use for relatively inexperienced protein biochemists, however there are a few considerations to bear in mind.
Caution about overfilling collection plates
The 48-well collection plate itself only holds 5 mL per well while each 96-well only holds 2 mL. This needs to be kept in mind when adding buffer and running sample through the column as there is the risk of overfilling the wells. In particular, care needs to be t...

Protein purification techniques to achieve milligram quantities of purified proteins are imperative to their characterization and analysis, especially for biophysical methods such as NMR. Protein purification is also central across other areas of study such as drug discovery processes and protein-protein interaction studies; however, achieving such quantities of pure protein can become a bottleneck for these techniques1,2,3. The principal method for protein purification is chromatography, which includes a variety of methods that rely on the individual characteristics of proteins and their tags. In affinity chromatography, proteins have an additional protein or peptide motif that works as a tag that has an affinity for a certain substrate on the chromatography resin4. The most common affinity method is immobilized metal affinity chromatography (IMAC) using His-tagged proteins, whereas another popular method is ion exchange chromatography that separates proteins based on their charge. For highest purity, a combination of affinity chromatography and ion exchange is frequently used together, usually requiring expensive lab equipment for high-throughput.
The evolving era of functional proteomics is fueling the demand for high-throughput techniques for purifying not singular proteins for specific analysis but large numbers of proteins simultaneously for comprehensive analysis and genome wide studies5. Immobilized metal affinity chromatography (IMAC) is one of the most widely used methods for high-throughput protein purification6,7 yet its automated systems are costly and unaffordable for smaller laboratories8. The more affordable plate-based alternatives that are currently available employ the use of accessible laboratory-based equipment, such as a vacuum. Although these methods are successful in improving the speed of purification, it can only achieve high-throughput purification on a smaller scale, only yielding protein in the microgram range. These limitations mean that the pre-packed 96 well filter plates (e.g., from GE Healthcare now owned by Cytiva) cannot be used before biophysical techniques9. Gravity chromatography is the most cost-efficient method of purification; however, setting up multiple columns is inconvenient and can be prone to error for multiple proteins.
A multi column plate adaptor (MCPA) has been developed and proven to successfully and conveniently run parallel affinity chromatography columns at once to purify His-tagged yeast SH3 domains10. The MCPA offers a cost-efficient high-throughput purification method that does not depend on costly instrumentation. Its flexible design can effectively purify milligrams of protein by multiple affinity chromatography columns under gravity or vacuum manifold. Furthermore, resin type, volume, and other parameters can be adjusted for each individual column for faster optimization. This study demonstrates that ion exchange chromatography by the MCPA can be used in conjunction with affinity chromatography by the MCPA to enhance the purification of the Abp1 SH3 domain. Additionally, up to 24 different proteins can be separated in parallel using these methods.

<table><tbody><tr><td>2 mL/ well collection plate</td><td>Agilent technologies</td><td>201240-100</td><td></td></tr><tr><td>5 mL/ well collection plate</td><td>Agilent technologies</td><td>201238-100</td><td></td></tr><tr><td>12 mL chromatography columns</td><td>Bio-Rad</td><td>7311550</td><td></td></tr><tr><td>96 well long drip plate</td><td>Agilent technologies</td><td>200919-100</td><td>Come with 0.25 um filters which are to be removed.</td></tr><tr><td>96 well plate seal/mat</td><td>Agilent technologies</td><td>201158-100</td><td>Should be peirceable</td></tr><tr><td>His60 Ni Superflow Resin</td><td>Takara Bio</td><td>635660</td><td></td></tr><tr><td>HiTrap Q HP anion exchange column</td><td>GE Healthcare (Cytiva)</td><td>17115301</td><td></td></tr><tr><td>Lvis plate reader</td><td>BMG LABTECH</td><td></td><td>Compatible with FLUOstar Omega plate reader</td></tr><tr><td>Male leur plugs</td><td>Cole-Parmer</td><td>EW-45503-70</td><td></td></tr><tr><td>PlatePrep 96 well Vacuum Manifold Starter kit</td><td>Sigma-Aldrich</td><td>575650-U</td><td></td></tr><tr><td>Reservoir collection plate</td><td>Agilent technologies</td><td>201244-100</td><td></td></tr><tr><td>The Repeater Plus</td><td>Eppendorf</td><td>2226020</td><td>With 5 mL and 50 mL syringes</td></tr><tr><td>VACUSAFE vacuum</td><td>INTEGRA</td><td>158 320</td><td>The vacusafe vacuum has a vacuum range from 300 mBar to 600 mBar and a 4 L waste collection bottle</td></tr></tbody></table>

an economical and versatile high-throughput protein purification system using a multi-column plate adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

As an example, the MCPA has successfully purified 14 AbpSH3 mutants in denaturing conditions via Ni-NTA (Figure 2A). A small contaminant ~ 25 kDa can be seen, however the protein is still largely pure. This contaminant is believed to be YodA, a common co-purified protein found in E. coli11.&#160;Figure 2B shows the purification of 11 different SH3 domains under native conditions. The small contaminant seen in denaturing ...

Watch this Scientific Journal Video about An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter at JoVE.com

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

an-economical-versatile-high-throughput-protein-purification-system

Research

JoVE Journal

Biochemistry

4.2K Views.  University of Liverpool. Een economisch en veelzijdig eiwitzuiveringssysteem met hoge doorvoer met behulp van een multi-kolomplaatadapter. Hallo, eiwitzuivering is noodzakelijk voor de studie van de eiwitstructuur en -functie. En het wordt meestal gebruikt in combinatie met biofysische technieken.Dit is vooral belangrijk in het tijdperk van functionele proteomics die een eiwitproductie met hoge doorvoer vereisen. We veronderstelden dat een multi-kolom plaatadapter, de MCPA, meerdere chromatografiekolommen van ...

Video: An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Biology

Optimizing FPLC for Efficient Purification of His-Tagged FEN1 Protein

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

Author Spotlight: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.

The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Source: Pokhrel, A. et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video