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An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter
Chapters
Summary May 21st, 2021
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.
Transcript
An economical and versatile high throughput protein purification system using a multi column plate adapter. Hi, protein purification is imperative to the study of protein structure and function. And it's usually used in combination with biophysical techniques.
This is especially important in the era of functional proteomics that require a high-throughput protein production. We hypothesized that a multi-column plate adapter, the MCPA, can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. With this adapter, we have developed an economical and versatile method of protein purification that can be used under gravity or vacuum rival in the speed of an automated system.
Here we show this method for nickel affinity chromatography and ion exchange chromatography. Assemble the MCPA by placing a punctured sealing mat onto a long drip plate, then insert the desired number of columns into the holes of the sealing mat. Place an open collection plates in the base of the manifold and close with the top of the manifold.
Attach tubing and place the assembled MCPA with columns on the top. Pipet 1.2 mil of the nickel-NTA solution into the columns. Whilst pipetting, ensure that the beads remain fully mixed.
Switch on the pump and run the 20%ethanol through the columns and into the collection plate below. Turn off the pump once the liquid is run through. Dispose of the contents of the collection plate into a waste box.
Add three resin volumes of EDTA buffer to all the columns. Turn on the pump and run the liquid through. Now wash the columns with three resin volumes of 0.5 molar sodium hydroxide buffer.
Wash the columns with four resin volumes of 100 millimolar nickel sulfate, then 10 resin volumes of Mili-Q water. If a column is running slower, push the liquid through with the syringe plunger. Pour out the contents of the collection plates into a waste box.
Then wash the columns twice with four resin volumes of 10 millimolar imidazole wash buffer and empty the collection plates. If multiple samples are being purified, replace the open collection plates with a 48-well plates. With the vacuum off, load the lysates into the columns.
Use a thin plastic stirrer to gently mix the beads and the lysates in the column to maximize binding. Turn on the pump and run the liquid through. If a column becomes blocked, transfer the lysate resin mixture to a column with a fresh filter.
Freeze the collection plates to contain your flow-through. Replaced with an open collection plate and then wash the columns with nine resin volumes of 10 millimolar imidazole wash buffer. Repeat this step four or five times.
To avoid overflow, periodically empty the waste plates. Replace the open collection plates with a 96-well plate, ensuring A1 is in the top left corner. Pipet one resin volume of denaturing elution buffer into the columns.
Run the liquid through and check the collection plate to ensure that there is no contamination between the wells. For the next dilution, repeat the previous steps and collect in a fresh collection plate. Take a 50 microliter aliquot of each elution for the purity and concentration analysis.
Next, wash the columns with two milliliters of 20%ethanol. Add another two mil of 20%ethanol to the columns and use the fresh thin plastic stirrer to mix up the beads in the ethanol before transferring to a 50 mil tube for storage at four degrees Celsius. Assemble the MCPA in vacuum manifold with an open collection plate and syringe plungers in all of the columns.
Remove all of the syringe plungers from the front row. Remove the rubber gasket from a five mil syringe plunger and then pierce a hole in the center. Then push the bottom of an open column through the punctured rubber gasket.
Repeat these steps and insert into the front row of columns. Ensure that the Q sepharose beads are fully mixed. And with the blue pipette tip that is cut two centimeters from the bottom, pipet 800 microliters of the Q sepharose beads into the open columns.
Once the beads have settled to the bottom of the columns, switch on the vacuum pump enough to run the 20%ethanol through. Wash the columns twice with two mil of 10 millimolar Tris. Turn off the pump just before the Tris buffer has run through to prevent the resin drying out.
Runoff can be discarded and replace with a 48-well collection plates. Transfer all samples to be purified to the Q Sepharose columns in the first row and use a thin plastic stirrer to mix the samples and the beads for around two minutes before turning on the vacuum pump. Store or freeze your flow-through for later analysis.
With an open collection plate, wash the columns twice with two mil of 10 millimolar Tris. Replace the open collection plates with the 96-well plate. The first elution fraction can be collected in the first row or in the second row.
To elute in the second row, remove the syringe plungers from these positions. Move the Q Sepharose columns into these positions and then place syringe plungers into the open columns in the first row. Pipet one milliliter of the first salt concentrations into the Q Sepharose columns.
Turn on the pump and collect the elution. To collect the next elution, remove the syringe plungers from the next positions, move the Q Sepharose columns into these positions and then replace the plungers in the previous positions. Repeat these steps for each successive salt concentration used to elute.
Ensure that a new collection plate is used for every four elutions and that every plate is labeled and stored. With an open collection plates, wash the columns with two mil of four molar sodium chloride, 10 millimolar Tris. Pipet two mil of 20%ethanol and run this through until it is just above the beads and then seal the columns for storage.
As an example, the MCPA has successfully purified 14 AbpSH3 mutants in denaturing conditions by a nickel-NTA. A small contaminant can be seen at 25 kilodaltons, though the protein is still largely pure. The small contaminants seen in denaturing conditions is removed in native conditions.
This was shown when 11 different SH3 domains were purified. This shows that the MCPA can be used for comparison of purification conditions. AbpSH3 can be separated from the majority of contaminants via ion exchange purification from lysates.
Good yields of considerably pure AbpSH3 protein were recovered with various higher molecular weights contaminants. Purification of samples post-nickel-NTA using ion exchange with the MCPA has successfully removed these high-weight contaminants. Though there is still a slight contamination, the fractions are still largely pure and have yielded good biophysical data using NMR and thermal chemical denaturation assays.
In conclusion, our results show that our hypothesis is correct and that the MCPA can successfully purify milligram quantities of proteins using different chromatography techniques. The MCPA system can be configured in multiple ways within the same runs to optimize purification conditions. The setup is simple, cheap, and easy to train inexperienced users, especially in labs that do not routinely purify proteins.
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