Products
rProtein A Agarose beads
- SPECIFICATIONS
- FIGURES
- CONDITIONS
- Catalog No.
K126
- Size
10mL/50mL/100mL
- Storage
2-8°C for 24 months
- Introduction
Protein A is derived from a strain of Staphylococcus aureus and contains five regions that bind to the Fc region of IgG. As an affinity ligand, protein A is coupled to Sepharose so that these regions are free to bind IgG. One molecule of protein A can bind at least two molecules of IgG.
The binding strength of protein A for IgG depends on the source species of the immunoglobulin as well as the subclass of IgG (see Table 1). The dynamic binding capacity depends on the binding strength and also on several other factors, such as flow rate during sample application.
Although IgG is the major reactive human immunoglobulin, some other types have also been demonstrated to bind to protein A. Interaction takes place with human colostral IgA as well as human myeloma IgA2 but not IgA1. Some human monoclonal IgMs and some IgMs from normal and macroglobulinaemic sera can bind to protein A.
Leakage of ligands from an affinity medium is always a possibility, especially if harsh elution conditions are used. The multi-point attachment of protein A to sepharose results in very low leakage levels over a wide range of elution conditions.
Table 1. Binding characteristics of different immunoglobulins (Ig).
Species
Subclass
ProteinAbinding
Species
Subclass
ProteinAbinding
Human
Total Ig
S
Goat
IgG2
S
IgG1, 2, 4
S
Sheep
Total Ig
W
IgG3
W
IgG1
W
IgD
N
IgG2
S
IgA, IgM
W
Cow
Total Ig
W
Fab
W
IgG1
W
ScFv
W
IgG2
S
Mouse
Total Ig
S
Horse
Total Ig
W
IgG1
W
IgG(ab)
W
IgG2a, 2b, 3
S
IgG(c)
W
IgM
N
IgG(T)
N
Rat
Total Ig
W
Rabbit
Total Ig
S
IgG1
W
Dog
Total Ig
S
IgG2a
N
Cat
Total Ig
S
IgG2b
N
Pig
Total Ig
S
IgG2c
S
Guinea pig
Total Ig
S
Goat
Total Ig
W
Chicken
Total Ig
N
IgG1
W
*Native Protein A and protein A differ in their binding to Ig classes from different species and subclasses. S: strong binding; M: medium binding; W: weak binding; N: no binding.
- Product Characteristics
Parameter
Description
Matrix
High-rigidity agarose
Ligand
Alkali-resistant recombinant Protein A
Mean Particle Size
70 μm
Dynamic Binding Capacity(6 min)
65-80 mg h-IgG/mL wet gel
Maximum Flow Rate(25℃)
300 cm/h
Ligand Leakage
<10 ng mL
Pressure Tolerance
0.3MPa(3 bar)
CIP Conditions
0.1–0.5 M NaOH, pH 2-14
Storage Conditions
20% ethanol, 2–8°C
- Assay Protocol
Given that this product exhibits varying binding affinities for antibodies of different species and subtypes, and that the optimal buffer conditions required to maintain antibody bioactivity may differ, users are advised to perform method screening.
The following provides a reference purification protocol:
1. Equilibration
Use 5–10 column volumes (CV) of Buffer A to equilibrate the column until baseline is reached.
Buffer A Options:
20 mM phosphate buffer (PB) + 0.15 M NaCl, pH 7.0 OR 0.05 M borate + 4.0 M NaCl, pH 9.0
2. Sample Loading
Load an appropriate amount of sample.
For solid samples: Prepare in Buffer A.
For liquid samples: Dialyze against Buffer A before loading.
3. Washing
Wash the column with 5 CV of Buffer A until baseline is reached.
4. Elution
Perform a 10 CV linear gradient elution with Buffer B until 100% Buffer B is reached.
Buffer B Options:
20 mM citrate, pH 3.0 OR 0.1 M glycine, pH 3.0 OR 20 mM sodium acetate, pH 3.0
5. Neutralization
Collect the eluted product and immediately neutralize using a neutralization buffer (e.g., 1.0 M Tris-HCl, pH 9.0, or other suitable buffer) to stabilize the antibody pH.
6. Regeneration & Cleaning-in-Place (CIP)
After multiple uses, the column should be regenerated. Two recommended methods:
Method 1 (Acid Wash)
Wash the column with 3–5 CV of 0.1 M acetic acid (or 0.1 M acetic acid + 20% ethanol).
Rinse with buffer until neutral pH is reached before reuse.
Method 2 (Alkali Wash)
Wash the column with 3–5 CV of 0.1–0.5 M NaOH. Then Flush with 3–10 CV of pure water. Re-equilibrate with buffer until neutral pH is reached before reuse.