Protein A Sepharose

Details for Product No. ABIN412439, Supplier: Log in to see
Antigen
  • alpha protein
  • repB
Reactivity
Staphylococcus aureus (S. aureus)
Application
Immunoprecipitation (IP), Purification (Purif)
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Purpose Protein A Sepharose can be used for IgG purification and immunoprecipitation
Characteristics Protein A-Sepharose beads are prepared by covalently coupling recombinant Protein A to 6% cross-linked Sepharose beads. Protein A (ABIN412501) is a genetically engineered protein containing five IgG-binding regions of native Protein A. The cell wall binding region, albumin binding region and other non-specific regions have been eliminated from the recombinant Protein A to ensure the maximum specific IgG binding. The coupling technique is optimized to give a higher binding capacity for IgG & minimum leaching of recombinant Protein A. The IgG binding capacity of Protein A-Sepharose is ≥ 16 mg human or rabbit IgG per ml of wet beads. Protein A-Sepharose beads display high chemical & physical stability as well as high flow rate, hydrophilicity & high gel strength. It can be used for IgG purification and immunoprecipitation.
ProductDetails: Bead Ligand Protein A
ProductDetails: Bead Matrix Sepharose beads
ProductDetails: Bead Size 90 µm
Application Notes - Purification of monoclonal and polyclonal antibodies from culture media, serum, ascites fluid or hybridoma supernatants.
- Isolation of antibody/antigen complexes in immunoprecipitation experiments, since only the Fc region is involved in antibody binding andthe Fab region is available for binding antigen.
Comment

Protein A-Sepharose beads are prepared by covalently coupling recombinant Protein A to 6% cross-linked Sepharose beads. The coupling technique is optimized to give a higher binding capacity for IgG & minimum leaching of recombinant Protein A. The IgG binding capacity of Protein A-Sepharose is ≥ 16 mg human or rabbit IgG per ml of wet beads.
BINDING CAPACITY: Binding of IgG ≥ 16 mg human or rabbit IgG/ml Protein A-Sepharose.
FLOW RATE TESTED: 2.07 ml/min
USAGE: Reusable for up to 10 times without significant loss of binding capacity.

Assay Procedure

PROTOCOL EXAMPLE (ANTIBODY PURIFICATION):
1. Carefully pack the column avoiding air bubbles.
2. Equilibrate the column with 5X resin bed volume of Binding Buffer & allow the buffer to drain through the column. Do not let the resin bed dry.
3. Dilute serum sample with Binding Buffer (1:1 ratio).
4. Mix well the diluted serum sample. Make sure there are no bubbles in the sample solution.
5. Apply the diluted sample onto the column. Do not let the resin bed dry.
6. Collect the flow-through.
7. Reapply the flow-through to the column & collect the sample. Repeat 4 times.
8. Wash the column 4 – 5 times with 5X volume of Binding Buffer containing 0.5 M NaCl.
9. Wash the column 4 - 5 times with Binding Buffer.
10. Elute antibodies with Elution Buffer ~3-5X resin bed volume.
11. Collect fractions using micro centrifuge tube. Immediately neutralize the eluted fractions by adding 100 µl of 1 M Tris, pH 9.0 per ml of eluate.
12. Assay protein concentration by measuring the absorbance at 280 nm and combine the fractions with highest absorbance. 1 OD280 = 0.73 mg/ml IgG.
13. To regenerate/store column:
a. Wash with 5 volumes of Elution Buffer.
b. Wash with 5 volumes of distilled water.
c. Store column in 20 % Ethanol/H2O at 4°C.

Restrictions For Research Use only
Format Liquid
Buffer Supplied as 50% slurry in 20% Ethanol/H2O.
Binding Buffer: PBS/TBS/0.15 M sodium chloride in 50 mM sodium borate, pH 8.0
Elution Buffer: 0.1 M citric acid, pH 2.75
Handling Advice Do not freeze!
Storage 4 °C
Expiry Date 12 months
Product cited in: Sury, McShane, Hernandez-Miranda, Birchmeier, Selbach et al.: "Quantitative proteomics reveals dynamic interaction of c-Jun N-terminal kinase (JNK) with RNA transport granule proteins splicing factor proline- and glutamine-rich (Sfpq) and non-POU ..." in: Molecular & cellular proteomics : MCP, Vol. 14, Issue 1, pp. 50-65, 2015 (PubMed).

Preston, Rabbani, Binder, Moes, Magnani, Ernst: "Implications of the E-selectin S128R mutation for drug discovery." in: Glycobiology, Vol. 24, Issue 7, pp. 592-601, 2014 (PubMed).

Schram, Ganguly, No, Fang, Thong, Sweeney: "Regulation of MT1-MMP and MMP-2 by leptin in cardiac fibroblasts involves Rho/ROCK-dependent actin cytoskeletal reorganization and leads to enhanced cell migration." in: Endocrinology, Vol. 152, Issue 5, pp. 2037-47, 2011 (PubMed).

Arana-Argáez, Delgado-Rizo, Pizano-Martínez, Martínez-Garcia, Martín-Márquez, Muñoz-Gómez, Petri, Armendáriz-Borunda, Espinosa-Ramírez, Zúñiga-Tamayo, Herrera-Esparza, Vázquez-Del Mercado: "Inhibitors of MAPK pathway ERK1/2 or p38 prevent the IL-1{beta}-induced up-regulation of SRP72 autoantigen in Jurkat cells." in: The Journal of biological chemistry, Vol. 285, Issue 43, pp. 32824-33, 2010 (PubMed).

Yang, Pursell, Lu, Chang, Mercurio: "Regulation of beta 4-integrin expression by epigenetic modifications in the mammary gland and during the epithelial-to-mesenchymal transition." in: Journal of cell science, Vol. 122, Issue Pt 14, pp. 2473-80, 2009 (PubMed).