INSTRUCTIONS SwellGel Immobilized Protein A, 96-Well Filter Plate 89828 3747 N. Meridian Road P.O. Box 117 Rockford, IL 61105 1331w Number Description 89828 SwellGel Protein A, 96-Well Filter Plate, contains sufficient materials to prepare 96 samples Kit contents: SwellGel Protein A Discs*, 96 discs each containing 100 µl of dehydrated Protein A resin, capacity: each disc will bind > 2.0 mg of Human IgG 96-well Deep Well Filter Plate, 1 each 96-well Collection Plate, 1 each Storage: Upon arrival store at 4 C. Product is shipped on ice. Keep plate sealed. This product is guaranteed for one year from the date of purchase when handled and stored properly. Introduction Protein A is a bacterial cell wall component produced by several strains of Staphylcoccus aureus. Protein A has the ability to specifically bind to the Fc region of immunoglobulin molecules, and has been extensively used as an affinity support for the isolation of a wide variety of IgG molecules from several species of mammals. SwellGel Protein A in a 96-well format is suited for high-throughput purification of mammalian polyclonal antibodies and for antigen purification using immunoprecipitation. The dehydrated nature of SwellGel Technology provides the user increased ease of handling, speed, convenience, consistency, greater stability of chromatography media in a high-throughput format and customized loading of a 96-well plate. (Individual wells can be used without damaging unused wells.) Procedure Summary Total procedure time: 35-45 minutes 1. Bind samples containing mammalian IgG 2. Wash 2-4 times to release non-bound protein 3. Elute IgG or continue with immunoprecipitation protocol Additional Materials Required Suitable Binding/Wash Buffer: See Important Product Information Suitable Elution Buffer: See Important Product Information Collection Plates: Three to four 650 µl capacity 96-square well collection plates Two to three 350 µl capacity 96-well collection plates. Multi-channel Pipette (optional) Centrifuge: Beckman Avanti J-20I Series or equivalent *Patent pending 1
Swinging Bucket Rotor: Beckman JS-4.3 or equivalent Important Product Information Use a clarified (centrifuged or filtered) protein solution to ensure proper resin flow. Binding of IgG to Protein A will occur under a variety of buffer conditions. However, Pierce recommends sample be in Modified Dulbecco's PBS (mpbs; 8.0 mm sodium phosphate 2.0 mm potassium phosphate, 140 mm sodium chloride, 10 mm potassium chloride, ph 7.4; Product No. 28374) or equivalent buffer. For serum samples, ascites fluid or tissue culture supernatant, it is necessary to dilute samples 1:1 with mpbs or a suitable buffer of your choice prior to sample application to the Protein A resin to ensure that the proper ph and ionic concentration is maintained for optimal binding. A variety of Elution Buffers for Protein A/IgG complexes are available. Pierce recommends using ImmunoPure IgG Elution Buffer (Product No. 21004 or 21009) or 0.1 M glycine buffer, ph 2.5-3.0. If you are planning on utilizing the purified IgG or antigen material in functional applications and the sample is known to be intolerant of the ph conditions in the Elution Buffer (ph 2-3) you may want to utilize our ImmunoPure Gentle Ag/Ab Elution Buffer system (Product No. 21027 or 21013). When using ImmunoPure Gentle Ag/Ab Elution Buffer the Wash Buffer must be changed to a non-phosphate-based buffer such as 50 mm Tris, 150 mm NaCl, ph 7.5. This is essential to avoid precipitation of buffer salts. Pierce still recommends using mpbs as the Binding Buffer. Allow buffers and protein samples to warm to room temperature before use. If the entire 96-well plate is not used at one time, unused wells can be used at a later date. Seal both the top and bottom of the filter plate for future use. Used wells can be regenerated. See Troubleshooting section. Store at 4 C. Keep tightly sealed. Do not allow moisture to get inside the container. Do not freeze. Example Protocols and Procedures The following protocols are examples of applications for this product. Your specific application may require optimization. Note: Protein A does not have identical binding characteristics for each subclass of IgG. See the Pierce Catalog for binding capabilities of immunoglobulin proteins. Standard Protocol for Purification of IgG using SwellGel Immobilized Protein A in 96- Well Plate Format Additional Materials Required for Purification of IgG Variable speed centrifuge with swinging buck rotor and plate carriers or vacuum manifold. Additional 96-well collection plates. Suitable Binding/Wash Buffer. Pierce recommends mpbs. (See Important Product Information for details.) Suitable Elution Buffer compatible with down- stream applications. Pierce recommends using ImmunoPure IgG Elution Buffer. (See Important Product Information for details.) 2
Procedure 1. Place a collection plate under the filter plate to collect flow-through. Grasp both filter and collection plates and tap gently to ensure that all discs have settled on the bottom of the filter plate. Remove seal from top of the filter plate. 2. Dilute 10-100 µl immunoglobulin containing sample to 200 µl with binding buffer. Note: See Important Product Information section for details on sample preparation. 3. Apply 200 µl of diluted immunoglobulin sample directly to the center of the wells in the 96-well filter plate. Incubate for 10-15 minutes at room temperature with gentle shaking. 3a. Place the filter plate and collection plate unit in a 96-well plate carrier. Centrifuge at 2,000 x g for 3 minutes. The flow-through is collected in the collection plate. If flow-through is needed for subsequent analysis, seal and keep the collection plate. Note: See centrifuge rotor instruction manual for conversion of gravity (x g) to rpm if needed. Note: A proper balance must be used as a counterweight for all the centrifugation steps. 3b. If sample contains low amounts of IgG or you wish to maximize binding potential of protein A resin steps 3 and 4 can be repeated. Note: Maximum binding capacity is approximately 3.5 mg of Rabbit IgG per disc. 5. Place a 650 µl collection plate under the filter plate. Add 400 µl of Wash Buffer to each well. Centrifuge at 2,000 x g for 3 minutes. Repeat wash step 2-3 times. Replace or reuse collection plate as necessary. 6. Before eluting sample, verify that the resin has been thoroughly washed of residual proteins. The wash can be monitored for unbound protein by measuring absorbance at 280 nm (A 280 ), BCA Protein Assay, Coomassie Protein Assay or SDS- PAGE analysis. This step can be omitted with optimized protocols. Note: Use of UV transparent collection plates is required for monitoring A 280. 7. Add 200 µl Elution Buffer to each well. Pipette up and down 3-4 times to suspend resin. 8. Place 350 µl collection plate under the filter plate. Centrifuge at 2,000 x g for 3 minutes to collect eluent. Repeat elution procedure 2-3 times for a total of 3-4 elutions. Note: Purified IgG usually completely elutes in the first 2 fractions. Additional elution may be needed depending on the concentration of bound IgG. 9. If needed, raise the ph of the eluted protein fractions by addition of a suitable, more concentrated buffer such as 20 µl 0.5 M Tris buffer, ph 7.6. Note: See Important Product Information for more information on Elution Buffer ph. 10. Purified sample is now ready for subsequent screening procedures such as ELISA, A 280, SDS-PAGE, BCA Protein Assay or Coomassie Protein Assay. 11. Collection plates containing samples of interest may be covered with sealing film or pooled and transferred to a microcentrifuge tube. Samples can now be desalted or concentrated as needed for further processing. See the Troubleshooting section for modifying this procedure for use with a vacuum manifold or for resin regeneration. Standard Protocol for Immunoprecipitation Using SwellGel Immobilized Protein A in 96-well Filter Plate Additional Materials Required for Immunoprecipitation Variable speed centrifuge with swinging buck rotor and plate carriers or a vacuum manifold. Additional 96-well collection plates. 3
Suitable Binding/Wash Buffer. Pierce recommends Modified Dulbecco s Phosphate Buffer. (See Important Product Information for details.) Suitable Elution Buffer compatible with downstream applications. Pierce recommends using ImmunoPure IgG Elution Buffer (Product No.21004 ), ImmunoPure Gentle Ag/Ab Elution Buffer (Product No.21027) or 0.1 M glycine buffer, ph 2.5-3.0. (See Important Product Information for details.) A. Formation of Immune Complex 1. Add purified antigen-specific IgG to sample or cell lysate to form an immune complex. 2. Incubate the sample for 30 minutes at room temperature or at 4 C overnight. Note: The optimal incubation time, temperature and buffer condition for forming the immune complex will vary depending on the affinity of the antibody for the antigen and overall antibody and antigen concentration. General Recommendations: a. Total volume of immune complex to be added to each well should be 200 µl for proper binding to resin. However, 200 µl can be loaded multiple times to each Protein A disc if required. b. While the buffer used for forming the immune complex can vary, care should be taken to ensure it is compatible with Protein A binding of IgG. Pierce recommends using Modified Dulbecco s PBS (8.0 mm sodium phosphate, 2.0 mm potassium phosphate, 0.14 M sodium chloride, 0.01 M potassium chloride, ph 7.4) as a Binding/Wash buffer. More 1.0% NP-40 or similar detergent can be added to the buffer (to optimize immune complex formation and reduce nonspecific binding). c. Recommended quantity of antibody to use per well: 0.1 mg - 2.0 mg of purified antibody. Note: As an alternate procedure, purified antibody can be pre-bound to the SwellGel Immobilized Protein A disc as in the IgG Purification Protocol above. Antigen-containing samples can then be applied to the swelled resin and incubated in the 96-well plate. Remainder of procedure below will then be followed as written starting at step 3a of section B. Capture of Immune Complex. B. Capture of Immune Complex 1. Place a collection plate under the filter plate to collect flow-through. Grasp both filter and collection plates and tap gently to ensure that all discs have settled on the bottom of the filter. Remove seal from top of the filter plate. 2. Apply 200 µl of sample containing antibody/antigen immune complex directly to the center of the wells in the 96-well filter plate. Incubate for 10-15 minutes at room temperature with gentle shaking. 3a. Place the filter plate and collection plate unit in a 96-well plate rotor, centrifuge at 2,000 x g for 3 minutes. The flowthrough is collected in the collection plate. Seal and keep the collection plate until binding of the antibody complex is confirmed. Note: A balance plate must be used as a counterweight for all the centrifugation steps. 3b. For larger volumes of sample containing low levels of antigen and antibody, steps 2-3 can be repeated until entire sample is applied. 4. Place a 650 µl collection plate under the filter plate. Add 400 µl of Wash Buffer to each well. Centrifuge at 2,000 x g for 3 minutes. Repeat wash step 2-3 times. Replace or reuse collection plate as necessary. 5. Before proceeding to elution protocol verify that the resin has been thoroughly washed to avoid contamination from residual proteins. The wash can be monitored for unbound protein by measuring A 280, BCA Protein Assay, Coomassie Protein Assay or SDS-PAGE analysis. This step can be omitted with optimized protocols. Note: Use of UV transparent collection plates is required for monitoring A 280. C. Elution of Immunoprecipitated Antigen 1. Add 200 µl Elution Buffer to each well. Pipette up and down several times to suspend resin. 2. Place 350 µl collection plate under the filter plate. Centrifuge at 2,000 x g for 3 minutes to collect eluent. Repeat elution procedure 2-3 times for a total of 3-4 elutions. 4
Note: IgG and antigen usually completely elute in the first 2 fractions. Additional elution may be needed to depending on concentration of bound antigen. 3. If needed, raise the ph of the eluted protein fractions by addition of a suitable, more concentrated buffer such as 20 µl 0.5 M Tris ph 7.6. 4. Purified sample is now ready for subsequent screening procedures such as ELISA or SDS-PAGE. 5. Collection plates containing samples of interest may be covered with sealing film or pooled and transferred to a microcentrifuge tube. Samples can be desalted or concentrated as needed for further processing. See Vacuum Manifold Information for modifying this procedure for use with a vacuum manifold or for resin regeneration. Vacuum Manifold Information 1. Because there are many variables between manufacturers' systems, Pierce recommends that the customer optimize conditions for their particular vacuum system. 2. It is important to use a slow bleed to prevent resin bed cracking. Faster is not better, in this instance. Use gentle suction. Use the bleed valve to get a minimum amount of vacuum for best results. 3. The Whatman Polyfiltronics UniVac 3-7705-0102 vacuum manifold has been specifically designed for use with the filter plates in which SwellGel Immobilized Protein G Discs are packaged. Most other manifold designs are also compatible with the SwellGel Disc 96-Well Plate format. 4. If all 96 wells are not used simultaneously, cover the top of the plate with plastic wrap. Apply a vacuum to seal the wells. Poke a small hole in the plastic above the wells being used to allow airflow and the wells to drain. Resin Regeneration 1. Protein A resin may be regenerated after sample elution by washing each well used with 400 µl binding/wash buffer or 10 mm Tris, ph 7.5. Centrifuge at 2,000 x g for 3 minutes. Repeat wash step 2 times. 2. For short-term storage, add 400 µl binding/wash buffer to the beads. Seal both top and bottom of the filter plate. Store at 4 C. Beads may be regenerated 2 times without significant loss of binding capacity. Related Pierce Products Immunology: Number Description 21004 ImmunoPure IgG Elution Buffer 21020 ImmunoPure Gentle Ag/Ab Elution Buffer 89830 SwellGel Immobilized Protein G, 96-well Filter Plate 45335 Seize Primary Immunoprecipitation Kit 20398 ImmunoPure Immobilized Protein G 20333 ImmunoPure Immobilized Protein A 20510 ImmunoPure Immobilized Protein L Protein Assays: 23227 BCA Protein Assay Reagent Kit 23236 Coomassie Plus Protein Assay Reagent Kit Pierce Chemical Co. 2/2002. Printed in the U.S.A. 5