LIT153B 9/2/98 8:52 AM Page Cvr1 Econo-Pac® Protein A Kit Protein A Columns Instruction Manual Catalog Number 732-2020 732-2022 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
LIT153B 9/2/98 8:52 AM Page i Table of Contents Section 1 Econo-Pac Protein A Kit ..........................................1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 Introduction .........................................................................1 Kit Components...................................................................1 Additional Items Required ..................................................2 Buffer Preparation ...............................................................2 Sample Preparation.
LIT153B 9/2/98 8:52 AM Page 1 Section 1 Econo-Pac Protein A Kit 1.1 Introduction The Econo-Pac protein A kit is based upon Bio-Rad’s Affi-Gel® protein A MAPS® II methods and contins everything necessary to purify monoclonal antibodies from ascites or serum. Affi-Gel protein A agarose consists of purified protein A coupled to crosslinked agarose beads via chemically stable amide bonds.
LIT153B 9/2/98 8:52 AM Page 2 1.3 Additional Items Required Column rack Test tubes pH meter Mixer Balance Filters The Econo-Pac 10 (12 place) acrylic rack is ideal for holding the Econo-Pac columns. Other benchtop or lattice mount racks may also be used. General purpose tubes for fraction collection are recommended. A pH meter is required to check the pH of the binding and elution buffers after reconstitution. Standard laboratory magnetic stirrer and bar for buffer mixing.
LIT153B 9/2/98 8:52 AM Page 3 1.5 Sample Preparation Sample preparation is simplified by using the Econo-Pac 10DG columns. Each Econo-Pac 10DG desalting column can process up to 3 milliliters of ascites fluid and the columns can be re-used. Two Econo-Pac 10DG columns should be reserved for buffer exchange at the end of the protein A purification protocol. It is also recommended that one Econo-Pac 10DG desalting column be used for each (different) ascites to avoid cross contamination.
LIT153B 9/2/98 8:52 AM Page 4 4. Wash the column with 20 ml binding buffer. 5. Elute the IgG with 10 ml of elution buffer. Elute the column with an additional 20 ml of buffer to insure total removal IgG. In most cases, the majority of the IgG will elute in the first 10 ml. 6. Wash the column with 10 ml of regeneration buffer, followed by 10 ml of PBS containing 0.02% sodium azide for storage. 7.
LIT153B 9/2/98 8:52 AM Page 5 4. Shelf life to the Econo-Pac protein A kit: The shelf life of the Econo-Pac protein A column is 1 year at 4 °C. The unreconstituted buffers are good for at least 1 year when stored at room temperature. Reconstituted buffers can be stored at 4 °C for approximately 2 weeks. 5. Purification of IgG from serum or tissue culture fluids: In addition to ascites, serum and tissue culture samples are suitable as samples for the Econo-Pac protein A kit.
LIT153B 9/2/98 8:52 AM Page 6 pleted with only one column volume of buffer per step, making the total procedure fast and easy. 2.2 Buffer Considerations The affinity of IgG for protein A is not the same for all species. Most mouse IgG1 immunoglobulins have low affinity for protein A. For this reason, the patented MAPS II buffer system has been developed to optimize binding and recovery of mouse IgG1.
LIT153B 9/2/98 8:52 AM Page 7 5. Elute the IgG with 10 ml of elution buffer. Elute the column with an additional 20 ml of buffer to insure total removal of IgG. In most cases, the majority of the IgG will elute in the first 10 ml. Prolonged exposure to acid pH should be avoided. Neutralize immediately after elution. The Econo-Pac 10DG desalting columns can be used to rapidly neutralize, desalt and buffer exchange the eluate into a suitable buffer (e.g., PBS).
LIT153B 9/2/98 8:52 AM Page 8 Section 3 References 1. Ey, P. L., Prowse, S. J. and Jenkins, C. R., Immunochemistry, 15, 429 (1978). 2. Begbee, W. L., Vanderlaan, M., Fong, S. S. N. and Jensen, R. M., Mol. Immunol., 20, 153 (1983). Section 4 Ordering Information Catalog Number Product Description 732-2020 Econo-Pac Protein A Kit, includes one 2 ml AffiGel protein A prepacked Econo-Pac column, 4 Econo-Pac 10 DG desalting columns, binding, elution, and regeneration buffers.
LIT153B 9/2/98 8:52 AM Page Cvr2 Bio-Rad Laboratories Life Science Group U.S. (800) 4BIORAD • California Ph. (510) 741-1000 • New York Ph. (516) 756-2575 • Australia Ph. 02-805-5000 • Austria Ph. (1) 877 89 01 • Belgium Ph. 09-385 55 11 • Canada Ph. (905) 712-2771 • China Ph. (01) 2046622 • Denmark Ph. 45-39 17 99 47 • France Ph. (1) 49 60 68 34 • Germany Ph. 089 31884-0 • India Ph. 91-11-4610103 • Italy Ph. 02-21609 1 • Japan Ph. 03-3534-7665 • Hong Kong Ph. 7893300 • The Netherlands Ph.
