Protein 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 1
month with addition of 0.02% sodium azide (NaN3) to the storage buffer. Because this product can purify
IgG subclasses from several species of mammals (see above), customers can conjugate the purification
products they got with SepharoseTM4B beads to purify secondary antibody.
Note: 20% ethanol was contained as protection solution in this product, please wipe off the ethanol
Protein A Beads Specifications
Matrix: CNBr-activated SepharoseTM4FF
Beads concentration: 1-2 mg/ml
Coupling conditions of matrix: pH 7-9, 4°C to 25°C, 2-16 h
Binding capacity: 4-7 mg IgG per ml
Bead size range: 45–165 ?m
Mean bead size: 90 ?m
Bead structure: Highly cross-linked agarose, 4%
Max. flow rate: 4 ml/min/cm2
Recommended flow rate: 1-3 ml/min/cm2
Stability of the matrix: pH 3-11 (ligand dependent)
Storage: Store at 4? for frequent use, at -20? for at least one year.
A: 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% glycerol
B. Sample preparation
1. Dilute the serum with equilibration buffer A to ensure its content and pH closed to equilibration buffer
2. Centrifuge diluted serum supernatants to sediment debris.
3. Filter supernatants through 0.45?m filter.
1. 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, equilibrate
column 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 total
volume 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. If
necessary, 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 during
collection. Then, customers can test the related data of the antibody as their own requirements.
D. Re-equilibration and Storage
1. Add 5-10ml Elution buffer to column to elute thoroughly, then neutralizate the column with
Equilibration buffer A.
2. Wash the column bed with Storage buffer in 3-5 column volumes, seal the bottom of the column and
store at -20? for at least one year. For frequent use, an aliquot can be stored at 4ºC for 1 month with
addition of 0.02% sodium azide (NaN3) to the storage buffer.