Alkali-resistant affinity medium for IgG purification

Ab-Capcher ExTra™


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Background

Ab-Capcher ExTra is an alkali-resistant affinity medium for IgG purification, which is coupled with an alkali-resistant Protein A-derivative (Protein A-R28) designed for higher IgG-binding capacity than that of Ab-Capcher. The dynamic binding capacity of Ab-Capcher ExTra is apporoximately 70 mg human IgG/mL of medium at 100 cm/min. Ab-Capcher ExTra shows higher binding to mouse and rat IgGs exceeding Ab-Capcher. Since Pyrogen testing is carried out on an every lot of manufacturing, antibodies purified with Ab-Capcher ExTra are suitable for cell-based assay*.

 

*Recommended that endotoxin-free buffers are used for purification of IgG with Ab-Capcher ExTra.

PTN_P0032_1

 

Protein A-R28 is an alkali-tolerant IgG-binding protein derived from protein A, which is developed with ProteNova’s patent technology. Protein A-R28 strongly binds to various species and subclasses of IgG, compared with Protein A and G. The coupling to resin (Ab-Capcher) provides an alkali-washable unique affinity medium with high binding capacity for immunoglobulin, which is useful for purification of human, rabbit, and mouse IgGs including mouse IgG1.Ab-Aapcher is also useful for immuno-precipitation experiments.
Binding Characteristics of Ab-Capcher (ExTra)
Species Subclass Ab-Cap Protein A Protein G
Mouse IgG1
IgG2a
++++
+++++
+
++++
++
+
Rat IgG1
IgG2a
++++
-* 
-
-

+
+

Goat IgGs ++++ - +
Chicken IgY - - -
Human IgG +++++ ++++ ++
Rabbit IgG +++++ ++++ ++

*Connection has not confirmed.

Application

Rat/Mouse/Human IgG purification

Feature and Advantages

◊Dynamic binding capacity (100 cm/hr) is 70 mg/mL.
◊Reusable by washing with alkali.

Comparison of binding capacities
PTN_P0032_2
Static binding capacity
(Maximum binding capacity)
Excess of human polyclonal IgG was applied to the gel, shaked for 1 hr at RT, washed and eluted at pH 2.8. Amount of IgG in the elute was measured.
Dynamic binding capacity
(Applied to a column)
Human polyclonal IgG (3 mg/mL) was applied to a column (5x100 mm) at linear velocity of 100 cm/hr (0.33mL/min). DBC at 10% brealthrough was determined. 
PTN_P0032_3

Specifications

Gel matrix : 6% highly closslinked agarose
Average particle size : 35 μm
Ligand : Alkali-resistant Protein A-derivative (Protein A-R28)
Ligand-coupling : secondary amine, multiple-point attachent
Binding capacity  
     Static : 100 mg Human IgG/mL of medium
    20 mg Mouse IgG1/mL of medium (purified from mouse ascitic fluid)
     Dynamic** : 85 mg Human IgG/mL of medium (60 cm/hr) 
  70 mg Human IgG/mL of medium (100 cm/hr) 
Maximum linear velocity : 500 cm/hr 
Pecommended liner velocity : 50-150 cm/hr 
Washing conditions : 0.1-0.5 M NaOH
Pyrogen testing : Endotoxin negative (Gel-Clot Technique) 
Preservative : 20% Ethanol 
Storage  : 4-8°C 

**Determined at 10% breakthrough on a column (5x108 mm, 2.1 mL) with 3 mg human IgG/mL

Application Data

1. Purification of mouse IgG from low-concentration samples by repeated addition

Gel volume: 100 μL/column
Sample: Purified mouse IgG1 (0.1 mg/mL PBS) 10 mL
Repeats: 20 times 

 

PTN_P0032_4
As simulation of purification from cultured medium, mouse monoclonal IgG1 at low concentration was purified by repeated addition of the sample. According to the protocol of Ab-Rapid SPiN, 0.5 mL of purified mouse IgG1 (0.1 mg/mL) was added to a SPiN column set in a centrifugal machine, stand for 4 min with sometimes mixing and centrifuged. These steps were repeated for 20 times. Total 10 mL of samples was added to the column, washed and eluted at pH2.8. Recovery (%) with Ab-Capcher ExTra us higher than that og Ab-Capcher, indicating that increasing dispersity of the medium at smaller particle size of 35 μm, influences the recovery of IgG.

 

2. Purification of mouse IgG1 from ascitic fluid

Gel volume: 50 μL/column
Sample: 1 mL of mouse ascitic fluid, x2 diluted with PBS
Incubation with gel: 2 hrs

 

 

PTN_P0032_5
Mouse ascitic fluid was 2-fold  diluted with PBS, applied to gel and shaked for 2 hrs. After washing, IgG was eluted at pH 2.8. Ab-Capcher ExTra shows 5.1-fold higher amount of IgG binding than that of Protein G gel and 30% higher than that of Ab-Capcher.

 

3. Purification of rat IgGs from rat serum
PTN_P0032_6
Rat IgGs were purified from rat serum with approximately 95% recovery, showing approximately 20% higher than that of Ab-Capcher.
Product List

Product Name Cat# Quantity Price

Ab-Capcher ExTra DataSheet ,

PTN-P-003-2

2 ML ¥30,800
$411

Ab-Capcher ExTra DataSheet ,

PTN-P-003-10

10 ML ¥96,800
$1291

Ab-Rapid SPiN Ex DataSheet ,

PTN-P-014-10

10 COLUMN ¥23,400
$312

Ab-Rapid SPiN Ex DataSheet ,

PTN-P-014-5-1

2 COLUMN ¥59,800
$798

Ab-Rapid PuRe Ex DataSheet ,

PTN-P-015-2

2 COLUMN ¥20,900
$279

Ab-Rapid PuRe Ex DataSheet ,

PTN-P-015-10

10 COLUMN ¥83,600
$1115

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.


“Ab-Capcher ExTra™” also belongs to the following categories.