TITLE: ChroPack - Protein A
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 Antibodies with ChroPack Columns by DALEX Biotech.</span></div><div class = "text-block">With ChroPack Protein A columns you can quickly isolate monoclonal and  polyclonal antibodies from various sample materials. Antibodies bind  with high specificity to the recombinant protein A whose BSA binding  site has been removed. The ChroPack columns can be operated with  peristaltic pumps and common FPLC systems. The Luer-Lock connections  make changing the column particularly easy.</div><div class = "text-block">One-time autoclavable with  DALEX stabilizer for Protein A.</div><div class = "text-block">Each ml bed volume has a binding capacity of > 10 mg (tested  with polyclonal human Ig, the binding capacity varies according to  species and clones).</div><div class = "text-block">Up to 98 % purity in a single purification step.</div><div class = "text-block">No swelling/shrinking of the matrix in aqueous buffers.</div><div class = "text-block">High thermal stability, up to 15 minutes at 80 °C in aqueous buffers with neutral pH.</div><div class = "text-block">pH stability: short-term pH 2 - 8, long-term pH 3 - 8.</div><div class = "text-block">Suitable for all common low-pressure systems.</div></div>

[STEPS]
?. [What do you want to do?]
Do you want to purify antibodies or sanitize your column?Please choose below.
?. [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).
A dry column can be directly connected to the FPLC system without special precautions. The air will be forced out through the bottom outlet.
?. [Equilibration]
Equilibrate the column with 5 to 10 bed volumes wash buffer.
?. [Load and Wash]
Load the sample onto the column.
For optimal binding and purity, the pH of the sample should be 7.5 - 8.5 and should contain 150 - 300 mM NaCl. Ideally, the optimal pH value for binding must be determined.An easy way to achieve this is by adding 1/11 volume 0.5 M Tris, 2 M NaCl (pH 8.0) to your sample.If the yield is lower as expected try to increase the pH by adding phosphate buffer.  Removal of particulate matter from the sample by centrifugation or filtration (0.45 µm) is recommended.For slow or weak binding antibodies collect the flow through and apply it again.
?. [Load and Wash]
Wash the column with 10 to 20 bed volumes.
It is advisable to keep the flow-through and wash fractions for later analysis, e.g. SDS-PAGE.In case of unspecific hydrophobic and/or ionic interactions include up to 1 % Tween-20 and/or up to 0.5 M NaCl in the wash buffer.
?. [Elution]
Elute with 10 bed volumes elution buffer and collect fractions of 0.5 to 1 bed volumes.
?. [Elution]
Add neutralization solution (1 M TRIS HCl pH 8.5) to the eluate. Add 100 µl neutralization solution to 900 µl eluate.
?. [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, 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.
?. [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.
?. [Storage]
Flush with 5 bed volumes wash buffer (PBS) with 0.05 % (w/v) sodium azide. Disconnect the column, close and store at 4 °C or at RT.It is recommended to wait at least 12 hours before the next purification.