TITLE: ChroPlate - IMAC
AUTHORS: Alexandra Ehl, David Frommholz, Nadine Stefanczyk

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purification Guide for the Isolation of Histidine-tagged Proteins with ChroPlate Filtration Plates by DALEX Biotech.</span></div><div class = "text-block">ChroPlate-IMAC by DALEX Biotech offers a robust and convenient way to isolate polyhistidine-tagged proteins from bacterial, mamalian, and insect cell cultures.</div><div class = "text-block">Immobilized metal affinity chromatography (IMAC) is based on the interaction of the imidazole ring of histidine with transition metal ions immobilized on a solid support. Recombinant proteins with a 3 - 10 histidine fusion tag bind to these metal ions while unwanted proteins are removed by washing with excess binding buffer. Elution of the target protein is achieved by the addition of imidazole, EDTA or a low pH. </div><div class = "text-block">Easy and quick small scale fusion protein purification from various sources.</div><div class = "text-block">Low metal leaching from column.</div><div class = "text-block">Tolerates reducing reagents (e.g. DTT up to 10 mM).</div><div class = "text-block">The proprietary resin does not shrink or swell in aqueous buffers.</div><div class = "text-block">High pressure stability.</div><div class = "text-block">pH stability short term 2 - 8, long term 3 - 8.</div><div class = "text-block">Excellent thermal stability up to 15 minutes at 120 °C in aqueous buffers at neutral pH.</div><div class = "text-block">Can be dried for long term storage (80 °C for > 2 h).</div></div>

[STEPS]
?. [Equilibration]
Add 200 µl of nickel or cobalt solution to each well. Place the ChroPlate on a deep-well plate and centrifuge at 400 g for 60 seconds in a swing-out rotor.  For counterbalance of the centrifuge a dummy filter plate is included in the kit.
Apart from Ni2+ and Co2+, you can also use Cu2+ or Zn2+.The affinity of histidine towards the metal ions is in the order Cu > Ni > Zn > Co. However, the specificity is in the inverse order, i.e. copper will most likely result in best yields but with lower purity. In comparison, cobalt will result in a better purity but also lower yields.
?. [Equilibration]
Empty the deep-well plate and add 500 µl deionized water to each well of the ChroPlate. Centrifuge at 400 g for 2 minutes in a swing-out rotor.Repeat once more.
?. [Equilibration]
Empty the deep-well plate and add 500 µl wash buffer to each well of the ChroPlate. Centrifuge at 400 g for 2 minutes in a swing-out rotor.
?. [Load and Wash]
Place the ChroPlate onto a clean deep-well plate. Add up to 1.5 ml cleared lysate and centrifuge at 400 g for 2 - 10 minutes in a swing-out rotor.
It is advisable to keep the flow-through and wash fractions for later analysis, e.g. SDS-PAGE.Repeat the loading step when your sample volume is larger than 1.5 ml. This is particularly recommended when your target protein has low expression levels.The centrifugation time depends on the sample's volume and viscosity. Volumes larger than 1 ml and viscous samples e.g. serum usually require more than 5 minutes centrifugation time.
?. [Load and Wash]
Empty the deep-well plate or use a clean one, add 500 µl wash buffer to each well of the ChroPlate, and centrifuge at 400 g for 2 minutes in a swing-out rotor.Repeat once more.
For an increase in purity repeat this step a third time. However, the yield will be lowered.
?. [Elution]
Place the ChroPlate on a clean deep-well plate. Add 300 µl elution buffer to each well and centrifuge 2 minutes at 400 g in a swing-out rotor.Elute two more times into the same deep-well plate.
?. [Buffer Preparation]
Binding BufferAdjust to pH 8.0 with NaOHElution BufferAdjust to pH 8.0 with NaOH
[Sodium dihydrogen phosphate]
[Urea]
[Sodium chloride]
[Sodium dihydrogen phosphate]
[Urea]
[Sodium chloride]
[Imidazole]
?. [Bacterial Cell lysis]
Determine the weight of the frozen bacterial pellet and thaw on ice.Resuspend the pellet in binding buffer by pipetting. For every gram of pellet add 5 milliliters buffer.Add lysozyme and DNaseIAlternatively use ultrasonication according the instuctions of your instrument manufacturer.
[0.1mg/ml each]
?. [Bacterial Cell lysis]
Incubate for 30 minutes with gentle end-over-end mixing, stirring, or rocking at room temperature.If your target is known to undergo proteolytic degradation incubate at 4 - 8 °C for 60 minutes.or
22 °C
4 °C
?. [Bacterial Cell lysis]
Centrifuge the lysate for 30 minutes at >10.000 g at .Carefully transfer the supernatant to a fresh tube.
4 °C
?. [Sample Preparation]
Incubate for 15 minutes with gentle end-over-end mixing, stirring, or rocking at room temperature.If your target protein is known to undergo proteolytic degradation or rapid denaturation, incubate at 4 - 8 °C for 30 minutes.or
22 °C
4 °C
?. [Sample Preparation]
Centrifuge the lysate for 30 minutes at >10.000 g at .Carefully transfer the supernatant to a fresh tube.
4 °C
During centrifugation you can already proceed with the equilibration of filter plate.
?. [Solutions]
deionized waterCleaning solution 1: 6 M Guanidine chloride, 0.2 M Acetic acidCleaning solution 2: 1 % Sodium dodecyl sulfate (SDS)Cleaning solution 3: > 95 % EthanolCleaning solution 4: 0.1 M Ethylenediaminetetraacetic acid (EDTA)Cleaning solution 5: 0.2 M Acetic AcidMetal solution: 0.1 M Nickel(II) sulfate (or other metal ion of your choice) 20 % (v/v) Ethanol
?. [Wash and Clean]
Remove the bottom cap and drain any storage solution by centrifugation at 10.000 g for 1 minute.
?. [Wash and Clean]
?. [Wash and Clean]
?. [Wash and Clean]
?. [Wash and Clean]
?. [Wash and Clean]
Use 500 µl of each Cleaning solutions solution, one after another, with two 500 µl washes with deionized water in between each of the Cleaning solutions, i.e.: Add 500 µl of Cleaning solution 1, invert sharply 4-5 times, centrifuge 1 minute at 10.000 g, discard flow through. Add 500 µl of deionized water, invert sharply 4-5 times, centrifuge 1 minute at 10.000 g, discard flow through. Add 500 µl of deionized water, invert sharply 4-5 times, centrifuge 1 minute at 10.000 g, discard flow through. Add 500 µl of Cleaning solution 2, invert sharply 4-5 times, centrifuge 1 minute at 10.000 g, discard flow through. Add 500 µl of deionized water, invert sharply 4-5 times, centrifuge 1 minute at 10.000 g, discard flow through. ...
?. [Final Wash and Recharge]
Wash the columns three times with deionized water as in the previous step.Add 500 µl of the Metal solution and incubate for 5 minutes with end-over-end mixing.Centrifuge 1 minute at 10.000 g, discard the flow through.Wash two more times with water.
?. [Storage]
Close the bottom of the column, add 500 µl 20 % Ethanol, and close the lid and store at 4 - 8 °CAlternative for long-term storage:Dry the open (top and bottom) columns in an oven at 60 °C for at 2 hours or over night. Make sure the bottom stopper is completely dry, too. Put on the column's outlet, close the lid and store the column(s) closed at room temperature.
?. [How do you want to purify?]
Please choose below how you want to proceed.
?. [Equilibration]
Assemble your vacuum device according to the manufacturer's instructions.Add 200 µl nickel or cobalt solution to each well, place the ChroPlate on a waste receptacle and apply a vacuum of 100 mbar for 2 minutes.
Apart from Ni2+ and Co2+, you can also use Cu2+ or Zn2+.The affinity of histidine towards the metal ions is in the order Cu > Ni > Zn > Co. However, the specificity is in the inverse order, i.e. copper will most likely result in best yields but with lower purity. In comparison, cobalt will result in a better purity but also lower yields.
?. [Equilibration]
Add 500 µl deionized water to each well, place the ChroPlate on a waste receptacle and apply a vacuum of 100 mbar for 2 minutes.Repeat once more.
?. [Equilibration]
Add 500 µl wash buffer to each well, place the ChroPlate on a waste receptacle and apply a vacuum of 100 mbar for 2 minutes.
?. [Load and Wash]
Add up to 1.5 ml cleared lysate to each well and apply a vacuum of 100 mbar for 2 minutes.
If you want to collect the flow through install a deep-well plate instead of the waste receptacle on the vacuum station.Repeat the loading step when your sample volume is larger than 1.5 ml. This is particularly recommended when your target protein has low expression levels.The vacuum time depends on the sample's volume and viscosity. Increase the vacuum or the time accordingly.
?. [Load and Wash]
Add 500 µl wash buffer to each well and apply a vacuum of 100 mbar for 2 minutes.Repeat once more.
If you want to collect the wash fraction, do not forget to mount a deep-well plate under the ChroPlate.For higher purities wash a thrid time. However, the yield will decrease.
?. [Elution ]
Place the ChroPlate on a clean deep-well plate, add 100 µl elution buffer to each well, and apply a vacuum of 100 mbar for 2 minutes.Elute two more times into the same deep-well plate.
?. [How do you want to purifiy?]
Please choose below how you want to proceed.
?. [Equilibration]
Add 200 µl of nickel or cobalt solution to each well. Place the ChroPlate on a deep-well plate and centrifuge at 1000 g for 60 seconds in a swing-out rotor.
Apart form Ni2+ and Co2+, you can also use Cu2+ or Zn2+.The affinity of histidine towards the metal ions is in the order Cu > Ni > Zn > Co. However, the specificity is in the invers order, i.e. copper will most likely result in best yields but with lower purity. In comparison, cobalt will result in a better purity but also lower yields.
?. [Equilibration]
Empty the deep-well plate and add 500 µl water to each well of the ChroPlate. Centrifuge at 1000 g for 5 minutes in a swing-out rotor.Repeat once more.
?. [Equilibration]
Empty the deep-well plate and add 500 µl binding buffer to each well of the ChroPlate. Centrifuge at 1000 g for 5 minutes in a swing-out rotor.
?. [Load and Wash]
Place the ChroPlate onto a clean deep-well plate. Add up to 1.5 ml cleared lysate, centrifuge at 1000 g for 5 - 10 minutes in a swing-out rotor.
Repeat the loading step when your sample volume is larger the 1.5 ml. This is particularly recommended when your target protein has low expression levels.The centrifugation time depends on the sample's volume and viscosity. Volumes larger than 1 ml and viscous samples e.g. serum require usually require more than 5 minutes centrifugation time.
?. [Load and Wash]
Empty the deep-well plate or use a clean one, add 500 µl binding buffer to each well of the ChroPlate, and centrifuge at 1000 g for 5 minutes in a swing-out rotor.Repeat once more.
?. [Elution]
Place the ChroPlate on a clean deep-well plate, add 200 µl elution buffer to each well of the ChroPlate, and centrifuge at 1000 g for 5 minutes in a swing-out rotor.Repeat once more.
?. [Equilibration]
Assemble your vaccum device according to the manufacturer's instructions.Add 200 µl metal solution to each well, place the ChroPlate on a waste recepticle and apply a vacuum of 100 mBar for 2 minutes.
Apart form Ni2+ and Co2+, you can also use Cu2+ or Zn2+.The affinity of histidine towards the metal ions is in the order Cu > Ni > Zn > Co. However, the specificity is in the invers order, i.e. copper will most likely result in best yields but with lower purity. In comparison, cobalt will result in a better purity but also lower yields.
?. [Equilibration]
Add 500 µl water to each well, place the ChroPlate on a waste recepticle and apply a vacuum of 100 mBar for 2 minutes.
?. [Equilibration]
Add 500 µl binding buffer to each well, place the ChroPlate on a waste recepticle and apply a vacuum of 100 mBar for 2 minutes.
?. [Load and Wash]
Add up to 1.5 ml cleared lysate to each well and apply a vacuum of 100 mBar for 2 minutes.
If you want to collect the flow through install a deep-well plate instead of the waste recepticle on the vacuum station.Repeat the loading step when your sample volume is larger the 1.5 ml. This is particularly recommended when your target protein has low expression levels.The vacuum time depends on the sample's volume and viscosity. Increase the vacuum or the time accordingly.
?. [Load and Wash]
Add 500 µl binding buffer to each well and apply a vacuum of 100 mBar for 2 minutes.Repeat once more
If you want to collect the wash fraction, do not forget to mount a deep-well plate under the ChroPlate.For higher purities wash a third time. However, the yield will decrease.
?. [Elution]
Place the ChroPlate on a clean deep-well plate, add 200 µl elution buffer to each well of the ChroPlate, and apply a vacuum of 100 mBar for 2 minutes.Repeat once more.
?. [What do you want to do?]
How do you want to purify your protein?Please choose below.
?. [Buffer Preparation]
Wash BufferAdjust to pH 8.0 with NaOHElution BufferAdjust to pH 8.0 with NaOH
[Sodium dihydrogen phosphate]
[Urea]
[Sodium chloride]
[Sodium dihydrogen phosphate]
[Urea]
[Sodium chloride]
[Imidazole]
Buffers containing urea are not stable at room temperature. Prepare freshly, use the buffers on the same day or store in appropriately sized aliquots at -20 °C for no longer than two months. Do not freeze and thaw the buffer more than once.
?. [Sample Preparation]
Determine the weight of the frozen pellet and thaw it on ice.Resuspend the pellet in wash buffer by pipetting. For every gram of pellet add 3 - 5 milliliters buffer.Add lysozyme and DNaseI (not included in the kit)Alternatively use ultrasonication according to the instructions of your instrument manufacturer.
[0.1mg/ml each]
Pre-chill an appropriate centrifuge to 4 °C.
?. [Sample Preparation]
Incubate for 30 minutes with gentle end-over-end mixing, stirring, or rocking at room temperature.If your target is known to undergo proteolytic degradation incubate at 4 - 8 °C for 60 minutes.or
22 °C
4 °C
?. [Sample Preparation]
Centrifuge the lysate for 30 minutes at >10.000 g at .Carefully transfer the supernatant to a fresh tube.
4 °C
During centrifugation you can already proceed with the equilibration of the filter plate.
?. [How do you want to purify?]
Please choose below how you want to proceed.
?. [Equilibration]
Add 200 µl of nickel or cobalt solution to each well. Place the ChroPlate on a deep-well plate and centrifuge at 400 g for 60 seconds in a swing-out rotor. For counterbalance of the centrifuge a dummy filter plate is included in the kit.
Apart from Ni2+ and Co2+, you can also use Cu2+ or Zn2+.The affinity of histidine towards the metal ions is in the order Cu > Ni > Zn > Co. However, the specificity is in the invers order, i.e. copper will most likely result in best yields but with lower purity. In comparison, cobalt will result in a better purity but also lower yields.
?. [Equilibration]
Empty the deep-well plate and add 500 µl deionized water to each well of the ChroPlate. Centrifuge at 400 g for 2 minutes in a swing-out rotor.Repeat once more.
?. [Equilibration]
Empty the deep-well plate and add 500 µl wash buffer to each well of the ChroPlate. Centrifuge at 400 g for 2 minutes in a swing-out rotor.
?. [Load and Wash]
Place the ChroPlate onto a clean deep-well plate. Add up to 1.5 ml cleared lysate, centrifuge at 400 g for 2 - 10 minutes in a swing-out rotor.
It is advisable to keep the flow-through and wash fractions for later analysis, e.g. SDS-PAGE.Repeat the loading step when your sample volume is larger than 1.5 ml. This is particularly recommended when your target protein has low expression levels.The centrifugation time depends on the sample's volume and viscosity. Volumes larger than 1 ml and viscous samples e.g. serum usually require more than 5 minutes centrifugation time.
?. [Load and Wash]
Empty the deep-well plate or use a clean one, add 500 µl wash buffer to each well of the ChroPlate, and centrifuge at 400 g for 2 minutes in a swing-out rotor.Repeat once more.
For an increase in purity repeat this step a third time. However, the yield will be lowered.
?. [Elution]
Place the ChroPlate on a clean deep-well plate. Add 300 µl elution buffer to each well and centrifuge 5 minutes at 400 g in a swing-out rotor.Repeat two more times.
?. [Equilibration]
Assemble your vacuum device according to the manufacturer's instructions.Add 200 µl nickel or cobalt solution to each well, place the ChroPlate on a waste receptacle and apply a vacuum of 100 mbar for 2 minutes.
Apart from Ni2+ and Co2+, you can also use Cu2+ or Zn2+.The affinity of histidine towards the metal ions is in the order Cu > Ni > Zn > Co. However, the specificity is in the invers order, i.e. copper will most likely result in best yields but with lower purity. In comparison, cobalt will result in a better purity but also lower yields.
?. [Equilibration]
Add 500 µl deionized water to each well, place the ChroPlate on a waste receptacle and apply a vacuum of 100 mbar for 2 minutes.Repeat once more.
?. [Equilibration]
Add 500 µl wash buffer to each well, place the ChroPlate on a waste receptacle and apply a vacuum of 100 mbar for 2 minutes.
?. [Load and Wash]
Add up to 1.5 ml cleared lysate to each well and apply a vacuum of 100 mbar for 2 minutes.
If you want to collect the flow through install a deep-well plate instead of the waste receptacle on the vacuum station.Repeat the loading step when your sample volume is larger than 1.5 ml. This is particularly recommended when your target protein has low expression levels.The vacuum time depends on the sample's volume and viscosity. Increase the vacuum or the time accordingly.
?. [Load and Wash]
Add 500 µl wash buffer to each well and apply a vacuum of 100 mbar for 2 minutes.Repeat once more.
If you want to collect the wash fraction, do not forget to mount a deep-well plate under the ChroPlate.For higher purities wash a thrid time. However, the yield will decrease.
?. [Elution]
Place the ChroPlate on a clean deep-well plate. Add 100 µl elution buffer to each well apply a vacuum of 100 mbar for 2 minutes.Repeat two more times.
?. [Sample Preparation]
Determine the weight of the frozen bacterial pellet and thaw it at room temperature.Resuspend the pellet in wash buffer by pipetting. For every gram of pellet add 3 - 5 milliliters buffer.Add lysozyme and DNaseI (not included in the kit) Alternatively, use ultrasonication according to the instructions of your instrument's manufacturer and skip the next step.
[0.1mg/ml each]
Pre-chill an appropriate centrifuge to 4 °C.