Community College of Rhode Island
400 East Avenue
Warwick, RI 02886
Document Number: 1.0
Revision Number: 0
Effective Date: 072207
Page 1 of 2
Title: Preparation of Protein A Chromatography Column
Approvals:
Preparer: ______Josephine Pino___________Date____072207____
Reviewer:_____ Christina Befumo_________Date____072207____
Reviewer:_____Tom Whitfield____________Date____072207____
1. Purpose:
1.1 To prepare a Protein A chromatography column for use.
2. Scope:
2.1. Applies to preparing a column for use in purifying IgG from serum using the
BioLogic LP System.
3. Responsibilites
3.1. It is the responsibility of the course instructor/lab assistant to ensure that this SOP
is performed as described and to update the procedure when necessary.
3.2. It ist he repsonsibility of the students/technicians to follow the SOP as described
and to infrom the instrucotr about any deviations or problems that may occur while
performing.the procedure.
4. References:
4.1 Affi-Gel Protein A agarose Manufacturer’s Instructions. (Bio-rad)
4.2. Binding Buffer and phosphate buffered saline/0.05% NaN3 (“PBS/Azide”) SOPs
5. Definitions:
5.1. Protein A - a protein from Staphylococcus aureus, which binds with high
specificity to the Fc region of IgG.
6. Precautions:
6.1. If PBS/Azide is used for storage, care must be taken and gloves and lab coat worn
when working with it. It is toxic if swallowed.
7. Materials:
7.1. Biorad 1x5cm Econo-column and accessories.
7.2. Affi-Gel Protein A agarose.
7.3. Phosphate Buffered Saline (PBS) with 0.05% Sodium Azide
7.4. Binding Buffer (3.2 M NaCl in 1.6 M Gycine, pH 9, filter-sterilized
7.5. beaker for collecting waste
7.6 50mL serological pipet (large bore size will protect gel)
7.7. Parafilm.
8. Position Column:
8.1. Attach Luer fitting-drain to bottom of the empty column.
8.2. Place column in a holder on a ring stand with a waste collection beaker
underneath.
8.3. Close valve.
8.4. Using a wide bore clean serological pipet, transfer approximately 3 mL of slurry
(this will yield approximately 1.5mL column volume) into the column.
8.5. Open the valve to allow liquid to drain out into the waste container.
Community College of Rhode Island
400 East Avenue
Warwick, RI 02886
Document Number: 1.0
Revision Number: 0
Effective Date: 072207
Page 2 of 2
8.6. If you plan to store more than one day, substitute binding buffer with PBS/Azide
in steps 8.6 -8.8.. Proceed as follows:
When the level of liquid is approximately 1cm above the level of the gel, gently
dispense 5mL of binding buffer to fill the column with a pasteur pipette. Allow the
buffer to flow down the side of the funnel top of the column.
8.7. Allow most of the binding buffer to drain out of the column. As it drains out, the
gel will settle. Do not disturb during this time to ensure that the level of the gel will be
horizontal and even.
8.8. Close the valve when the level of binding buffer is approximately 2 cm above the
gel. Confirm that the gel has settled completely leaving a column bed that is flat and
even. If the gel is still settling, gently add more buffer and allow it to drain until the
buffer is 2cm above the gel and settling is complete.
8.9. Cap the top of the column. Remove valve from the bottom and cap bottom.
Wrap each end with parafilm.
8.10. Keep the column upright at 4 C until use if not using immediately.
9. Attachments: N/A