TITLE: ChroPack - IMAC
AUTHORS: David Frommholz, Alexandra Raftery, 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 ChroPack Columns by DALEX Biotech.</span></div><div class = "text-block">
ChroPack-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 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>

[STEPS]
?. How do you want to purify your protein? Do you want to prepare your column for reuse or sanitize it?Please choose below.
?. [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.
?. [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 to a concentration of 0.1 mg/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
[at RT]
[at 4 °C]
?. [Sample Preparation]
Centrifuge: 10.000 34
4 °C
?. [Equilibration]
Connect the column to your FPLC system. Set the flow rate to 1 bed volume per minute. Wash the column with 5 volumes deionized water (bed volume is written on the column). Pump 0.5 column volumes of nickel or cobalt solution followed by another 5 bed volumes of deionized water.
A dry column can be directly connected to the FPLC system without special precautions. The air will be forced out through the bottom outlet.
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]
Equilibrate the column with 5 to 10 bed volumes wash buffer.
?. [Load and Wash]
Load the cleared lysate onto the column.
?. [Sample Preparation]
Filter the supernatant with a 0.45 µm filter.
?. [Load and Wash]
Wash the column for 10 to 20 bed volumes.
?. [Elution]
Elute with 10 bed volumes elution buffer and collect fractions of 0.5 to 1 bed volumes.
?. [Elution]
Wash the column successively with 5 column volumes of elution buffer, 5 column volumes wash buffer and 5 column volumes water. Then, wash with 5 column volumes 20 % ethanol or wash buffer containing 0.05 % (w/v) sodium azide.  Close the top lid and then the bottom stopper. Store at room temperature or at 4 - 8 °C
?. [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 to a concentration of 0.1 mg/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
[at RT]
[at 4 °C]
?. [Sample Preparation]
Centrifuge: 10.000 34
4 °C
?. [Sample Preparation]
Filter the supernatant with a 0.45 µm filter.
?. [Equilibration]
Connect the column to your FPLC system. Set the flow rate to 1 bed volume per minute. Wash the column with 5 volumes deionized water (bed volume is written on the column). Pump 0.5 column volumes of nickel or cobalt solution followed by another 5 bed volumes of deionized water.
A dry column can be directly connected to the FPLC system without special precautions. The air will be forced out through the bottom outlet.
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]
Equilibrate the column with 5 to 10 bed volumes wash buffer.
?. [Load and Wash]
Load the cleared lysate onto the column.
?. [Load and Wash]
Wash the column for 10 to 20 bed volumes.
?. [Elution]
Elute with 10 bed volumes elution buffer and collect fractions of 0.5 to 1 bed volumes.
?. [Elution]
Wash the column successively with 5 column volumes of elution buffer, 5 column volumes wash buffer and 5 column volumes water. Then, wash with 5 column volumes 20 % ethanol or wash buffer containing 0.05 % (w/v) sodium azide.  Close the top lid and then the bottom stopper. Store at room temperature or at 4 - 8 °C
?. [Sample Preparation]
Prepare Buffers:Wash Buffer:adjust to pH 8.0 with NaOHElution Buffer:adjust to pH 8.0 with NaOH
[Sodium dihydrogen phosphate]
[Urea]
[Sodium chloride]
[Sodium dihydrogen phosphate]
[Urea]
[Sodium chloride]
[Imidazole]
?. [Solutions]
Solutions needed: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]
Connect the column to your FPLC system and flush the column with 5 bed volumes water.
?. [Wash and Clean]
Pump 5 column volumes of cleaning solution 1 to 5, one after another, with 10 bed volume washes with deionized water in between each of the cleaning solutions.
?. [Recharge and Storage]
Wash the column one more time with 5 column volumes of deionized water. Then, pump 1 column volume of the metal solution.Wash with 10 column volumes of water
?. [Recharge and Storage]
Pump 5 bed volumes of 20 % (v/v) ethanol. Disconnect the column, close it and store either at 4°C or at RT.
?. [Sanitization]
Connect the column to your FPLC system and flush with 5 bed columes water, elution buffer and wash buffer (without urea). All steps are at a flow rate of 1 bed volume per minute.
?. [Sanitization]
Flush with 5 bed volumes sanitization solution, stop the pump and wait 1 hour.
?. [Sanitization]
Flush with 10 bed volumes water.
?. [Recharge and Storage]
Pump 0.5 bed volumes metal solution.
?. [Recharge and Storage]
Flush with 10 bed volumes water.
?. [Recharge and Storage]
Flush with 5 bed volumes 20 % (v/v) ethanol. Disconnect the column, close and store at 4 °C or at RT