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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purification Guide for the Isolation of Histidine-tagged Proteins with ChroDrip 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 protein 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]
If you start with a dry column, add 5 column volumes deionized water (bed volume is written on the column) and wait until it has drained. Add 0.5 column volumes of nickel or cobalt solution, let the solution drain and add another 5 column volumes of deionized water.When starting with a column which has been stored in storage solution, drain that solution first.
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 5 column volumes of wash buffer and wait until it has drained.
?. [Load and Wash]
Add the cleared lysate to the column and wait until it has drained.
Collect the flow through and wash fractions in separate tubes for later analysis, e.g. SDS-PAGE
?. [Load and Wash]
Add 5 column volumes of wash buffer, wait until it has drained and repeat once more.
For an increase in purity repeat this step a third time.
?. [Elution]
Add 0.75 column volumes of elution buffer to the column and let it drain.
This fraction does not contain the target protein. The small amount of elution buffer replaces most of the wash buffer in the column. This "pre-elution step" will result in a more concentrated eluate.
?. [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.
?. [Sample Preparation]
Determine the weight of the frozen bacterial pellet and thaw on ice.Resuspend the pellet in binding buffer by pipetting. For every gramm of pellet add 5 milliliters buffer.Add lysozyme and DNaseI (not included in the kit)Alternatively use ultrasonication according to the instuctions 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 column.
?. [Sample Preparation]
Determine the weight of the frozen bacterial pellet and thaw at room temperature.Resuspend the pellet in wash buffer by pipetting. For every gramm 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 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 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.
?. [Wash and Clean]
?. 
?. [Wash and Clean]
?. 
?. [Wash and Clean]
?. [Wash and Clean]
Use 5 column volumes of cleaning solution 1 to 5, one after another, with two 10 column volume washes with deionized water in between each of the cleaning solutions.
?. [Final Wash and Recharge]
Wash the column one more time with 5 column volumes of deionized water. Then, add 1 column volume of the metal solution, let it drain and wait for 5 minutes.Wash with 10 column volumes of water.
?. [Storage]
Add 10 column volumes 20 % ethanol or wash buffer (contains 0.05 % (w/v) sodium azide) and wait until half of it has drained. Then, attach the top and bottom stoppers.Alternative for long-term storage:After the final wash with water, 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 stopper, close the lid and store the column closed at room temperature.
?. [Elution]
Add 3 times 3 column volumes of elution buffer to the column. Wait inbetween the elution steps until the buffer has drained completely.
For more concentrated eluates, elute 8 times with one column volume and collect each fraction into a separate tube. Determine which fractions contain most of the protein and combine these.
?. [Equilibration]
If you start with a dry column, add 5 column volumes deionized water (bed volume is written on the column) and wait until it has drained. Add 0.5 column volumes of nickel or cobalt solution, let the solution drain and add another 5 column volumes of water.When starting with a column which has been stored in storage solution, drain that solution first.
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 5 column volumes of wash buffer and wait until it has drained.
?. [Load and Wash]
Add the cleared lysate to the column and wait until it has drained.
Collect the flow through and wash fractions in separate tubes for later analysis, e.g. SDS-PAGE
?. [Load and Wash]
Add 5 column volumes of wash buffer, wait until it has drained and repeat once more.
For an increase in purity repeat this step a third time.
?. [Elution]
Add 0.75 column volumes of elution buffer to the column and let it drain.
This fraction does not contain the target protein. The small amount of elution buffer replaces most of the binding buffer in the column. This "pre-elution step" will result in a more concentrated eluate.
?. [Elution]
Add 3 times 3 column volumes of elution buffer to the column. Wait inbetween the elution steps until the buffer has drained completely.
For more concentrated eluates, elute 8 times with one column volume and collect each fraction into a separate tube. Determine which fraction contain most of the protein and combine these.
?. [Sanitization]
After a purification: wash the column with 5 column volumes of elution buffer and 5 column volumes of wash buffer. If the column was stored in storage solution: let the buffer drain and wash with 5 column volumes of wash buffer.
?. [Sanitization]
Add 5 column volumes of sanitization solution, let 1 column volume drain and then close the column with the top and bottom cap. Incubate for one hour at RT.
?. [Sanitization]
Drain the sanitization solution and wash the column with 10 column volumes of deionized water.
?. [Cleaning and Storage]
Wash the column successively with 5 column volumes of elution buffer, 5 column volumes wash buffer and 5 column volumes water. Then, add 10 column volumes 20 % ethanol or wash buffer (contains 0.05 % (w/v) sodium azide). Wait until half of the buffer has drained. Close the top lid and then the bottom stopper. Store at room temperature or at 4 - 8 °C.Alternative 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. Close the column's outlet and the lid. Store at room temperature.
?. [Storage]
Add approximately 5 column volumes of 20 % ethanol or wash buffer (contains 0.05 % (w/v) sodium azide), let 1 column volume drain, close the top and bottom cap 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.
?. [Cleaning and Storage]
Wash the column successively with 5 column volumes of elution buffer, 5 column volumes wash buffer and 5 column volumes deionized water. Then, add 10 column volumes 20 % ethanol or wash buffer supplied in the kit (contains 0.05 % (w/v) sodium azide). Wait until half of the buffer is drained. Close the top lid and then the bottom stopper. Store at room temperature or at 4 - 8 °C.Alternative 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.