TITLE: ChroSpin - 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 ChroSpin Columns by DALEX Biotech.</span></div><div class = "text-block">ChroSpin-IMAC by DALEX Biotech offers a robust and convenient way to isolate polyhistidine-tagged proteins from bacterial, mammalian, 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 500 µl of nickel or cobalt solution, close the lid, and incubate 1 minute with end-over-end mixing.
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]
Remove the bottom lid.Centrifuge the column at 1000 g for 30 - 60 seconds.Discard the flow through.
?. [Equilibration]
Add 500 µl deionized water, close the lid, invert sharply 3 - 5 times, and centrifuge the column at 1000 g for 30 - 60 seconds. Discard the flow through.Repeat once more.
?. [Equilibration]
Add 500 µl wash buffer, close the lid, invert sharply 3 - 5 times, and centrifuge the column at 1000 g for 30 - 60 seconds.
?. [Load and Wash]
Place the column into a fresh collection tube. Add up to 500 µl cleared lysate, close the lid, and incubate 5 minutes with end-over-end mixing. Centrifuge the column at 1000 g for 30 - 60 seconds.
Repeat the loading step when your sample volume is larger than 500 µl. This is particularly recommended when your target protein has low expression levels.
It is advisable to keep the flow-through and wash fractions for later analysis, e.g. SDS-PAGE.
?. [Load and Wash]
Place the column into a fresh collection tube. Add 500 µl wash buffer, close the lid, and invert sharply 3 - 5 times. Centrifuge the column at 1000 g for 30 - 60 seconds and empty the collection tube.Repeat once more without changing the collection tube.
For increased purity, repeat the washing step up to 5 times.
?. [Elution]
Place the column into a fresh collection tube. Add 200 µl elution buffer to the column. Centrifuge the column at 1000 g for 30 - 60 seconds.Without changing the collection tube, repeat this elution step two more times.
?. [What do you want to do?]
How do you want to purify your protein? Do you want to prepare your column for reuse or sanitize it?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.
?. [Equilibration]
Add 500 µl of nickel or cobalt solution, close the lid, and incubate 1 minute with end-over-end mixing.
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]
Remove the bottom lid.Centrifuge the column at 1000 g for 30 - 60 seconds.Discard the flow through.
?. [Equilibration]
Add 500 µl deionized water, close the lid, invert sharply 3 - 5 times, and centrifuge the column at 1000 g for 30 - 60 seconds. Discard the flow through.Repeat once more.
?. [Equilibration]
Add 500 µl wash buffer, close the lid, invert sharply 3 - 5 times, and centrifuge the column at 1000 g for 30 - 60 seconds.
?. [Load and Wash]
Place the column into a fresh collection tube. Add up to 500 µl cleared lysate, close the lid, and incubate 5 minutes with end-over-end mixing. Centrifuge the column at 1000 g for 30 - 60 seconds.
Repeat the loading step when your sample volume is larger than 500 µl. This is particularly recommended when your target protein has low expression levels.
It is advisable to keep the flow-through and wash fractions for later analysis, e.g. SDS-PAGE.
?. [Load and Wash]
Place the column into a fresh collection tube. Add 500 µl wash buffer, close the column, and invert sharply 3 - 5 times. Centrifuge the column at 1000 g for 30 - 60 seconds and empty the collection tube. Repeat once more without changing the collection tube.
For increased purity, repeat the washing step up to 5 times.
?. [Elution]
Place the column into a fresh collection tube. Add 200 µl elution buffer to the column. Centrifuge the column at 1000 g for 30 - 60 seconds.Without changing the collection tube, repeat this elution step two more times.
?. [Sample Preparation]
Determine the weight of the frozen bacterial 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 protein is known to undergo proteolytic degradation incubate at 4 - 8 °C for 60 minutes.or
?. [Sample Preparation]
Centrifuge the lysate for 30 minutes at >10.000 g at .Carefully transfer the supernatant to a fresh tube.
During centrifugation you can already proceed with the equilibration of the spin column.
?. [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 instuctions of your instrument manufacturer and skip the next step.
[0.1mg/ml each]
Pre-chill an appropriate centrifuge to 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
?. [Sample Preparation]
Centrifuge the lysate for 30 minutes at >10.000 g at .Carefully transfer the supernatant to a fresh tube.
During centrifugation you can already proceed with the equilibration of the spin column.
?. [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 1000 g for 1 minute.
?. [Wash and Clean]
?. 
?. [Wash and Clean]
?. 
?. [Wash and Clean]
?. [Wash and Clean]
Use 500 µl of each cleaning 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 1000 g, discard flow through. Add 500 µl of deionized water, invert sharply 4-5 times, centrifuge 1 minute at 1000 g, discard flow through. Add 500 µl of deionized water, invert sharply 4-5 times, centrifuge 1 minute at 1000 g, discard flow through. Add 500 µl of cleaning solution 2, invert sharply 4-5 times, centrifuge 1 minute at 1000 g, discard flow through. Add 500 µl of deionized water, invert sharply 4-5 times, centrifuge 1 minute at 1000 g, discard flow through. ...
?. [Final Wash and Recharge]
Wash the column 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 1000 g, discard the flow through.Wash two more times with deionized water.
?. [Storage]
Close the bottom of the column, add 500 µl 20 % Ethanol or wash buffer (contains 0.05 % (w/v) sodium azide), and close the lid and store at 4 - 8 °CAlternative for long-term storage:Dry the open (top and bottom) column in an oven at 80 °C for at least 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 closed at room temperature.
?. [Sanitization]
After purification: wash the column with 500 µl elution buffer, centrifuge the column for 30 - 60 seconds at 1000 g, repeat with 500 µl wash buffer. If the column was stored before: remove the bottom cap and centrifuge the column for 30 - 60 seconds at 1000 g. Then add 500 µl of wash buffer and centrifuge the column for 30 - 60 seconds at 1000 g.
?. [Sanitization]
Close the bottom of the column and add 500 µl sanitization solution. Close the lid and incubate for one hour at RT.
?. [Sanitization]
Remove the bottom cap and centrifuge the column for 30 - 60 seconds at 1000 g.Wash three times with 500 µl of deionized water.
?. [Storage]
Close the bottom of the column, add 500 µl 20 % ethanol or wash buffer (contains 0.05 % (w/v) sodium azide). Close the lid and store at room temperature or at 4 - 8 °C.
?. [Cleaning and storage]
Wash the column by adding 500 µl elution buffer and centrifuge at 1000 g for 30 - 60 seconds. Repeat this step with 500 µl wash buffer and 500 µl deionized water. Then, close the bottom of the column, add 500 µl 20 % ethanol or wash buffer (contains 0.05 % (w/v) sodium azide). Close the lid and store at room temperature or at 4 - 8 °C.
?. [Cleaning and Storage]
Wash the column by adding 500 µl elution buffer and centrifuge at 1000 g for 30 - 60 seconds. Repeat this step with 500 µl wash buffer and 500 µl deionized water. Then, close the bottom of the column, add 500 µl 20 % ethanol or wash buffer supplied in the kit (contains 0.05 % (w/v) sodium azide). Close the lid and store at room temperature or at 4 - 8 °C.