Catalog #APB0001-5Size 5mlIntroductionProtein A binds to most human and mouse IgG subclasses (e.g. human IgG1, IgG2, IgG3, IgG4, IgA;mouse IgG1, IgG2a, IgG2b, IgG3). It also binds to rat IgG1, IgG2c; goat IgG1, IgG2; sheep IgG1, IgG2;cow IgG1, IgG2; horse IgG (ab), IgG(c). Protein A binds strongly to total IgG from rabbit, dog, cat, pig,guinea pig.This product can be used for 100-200 times. For frequent use, an aliquot can be stored at 4ºC for 1month with addition of 0.02% sodium azide (NaN3) to the storage buffer. Because this product can purifyIgG subclasses from several species of mammals (see above), customers can conjugate the purificationproducts they got with SepharoseTM 4B beads to purify secondary antibody.Note: 20% ethanol was contained as protection solution in this product, please wipe off the ethanolbefore use.Protein A Beads SpecificationsMatrix: CNBr-activated SepharoseTM 4FFBeads concentration: 1-2 mg/mlCoupling conditions of matrix: pH 7-9, 4°C to 25°C, 2-16 hBinding capacity: 4-7 mg IgG per mlBead size range: 45–165 μmMean bead size: 90 μmBead structure: Highly cross-linked agarose, 4%Max. flow rate: 4 ml/min/cm2Recommended flow rate: 1-3 ml/min/cm2Stability of the matrix: pH 3-11 (ligand dependent)Storage: Store at 4℃ for frequent use, at -20℃ for at least one year.
ProtocolA: Buffers preparation Equilibration buffer A: 1% Nacl+0.1% Na2HPO4, pH≈7.5 Equilibration buffer B: 1% CH3COONa adjusted pH to 5 by CH3COOH. Elution buffer: CH3COOH(pH =2~3) or 0.1mol Glycine Hydrochloride. Wash buffer: 1% Nacl+0.1% Na2HPO4, pH≈7.5 Storage buffer: 30% glycerolB. Sample preparation1. Dilute the serum with equilibration buffer A to ensure its content and pH closed to equilibration bufferA.2. Centrifuge diluted serum supernatants to sediment debris.3. Filter supernatants through 0.45μm filter.C. Affinity-purification1. Load the Protein A beads into the empty column.2. Wash column with Wash buffer in 3-5 column volumes to remove the glycerol, and then, equilibratecolumn by washing with Equilibration buffer A in 5-10 column volumes.3. Bring the sample to room temperature, and load it into the column by a syringe or a pump. The totalvolume of the sample applied is not critical in most cases.4. Load the sample into the column and collect the flow liquid, repeat this action for 3-5 times. Ifnecessary, repeat for more times, then deal with the collected liquid reasonably.5. Wash the column with Equilibration buffer B to remove other proteins.6. Elute with Elution buffer, collect the flow liquid (antibody), adjust its pH by saturated Na2CO3 duringcollection. Then, customers can test the related data of the antibody as their own requirements.D. Re-equilibration and Storage1. Add 5-10ml Elution buffer to column to elute thoroughly, then neutralizate the column withEquilibration buffer A.2. Wash the column bed with Storage buffer in 3-5 column volumes, seal the bottom of the column andstore at -20℃ for at least one year. For frequent use, an aliquot can be stored at 4ºC for 1 month withaddition of 0.02% sodium azide (NaN3) to the storage buffer.