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Product Name: Recombinant Protein G Sepharose (ProG Sepharose)
Catalog Number: NRPB02L, NRPB02S (prepacked column)
Packing Details: 10 ml, 100 ml, 1L, 1 ml (prepacked column), 2 ml (prepacked column), 5ml (prepacked column), 10ml (prepacked column)
Introduction:
Protein G is a bacterial cell wall protein expressing at the cell surface of some group C and group G Streptococcal strains. Protein G binds specifically to Fc regions of many mammalian immunoglobulins and is commonly used as affinity adsorbent to purify immunoglobulins (antibodies) and immunoglobulin subtypes from serum, hybridoma ascites, tissue culture supernatants and other biological fluids.
Column Characteristics:
Support
6% highly cross-linked spherical agarose
Ligand
recombinant protein G
Ligand Density (mg/ml)
>4
Particle Size (レm)
45~165
Flow Rate-Recommended/Maximum (cm/h)
100/750
pH Limits-Working/Cleaning
3~9/2~10
Maximum Operating Pressure (MPa)
0.3
Capacity
∶35 mg/ml IgG
Storage
4 oC to 8 oC in 20% ethanol
Some Typical Binding and Elution Conditions with Protein A Sepharose:
Species
Antibody Class
Protein G binding
pH value of binding buffer
pH value of elution buffer
Human
IgG1
+++
6.0~7.0
3.5~4.5
IgG2
+++
6.0~7.0
3.5~4.5
IgG3
+++
8.0~9.0
÷7.0
IgG4
+++
7.0~8.0
2.5~4.5
Cow
IgG2
+++
7.0~8.0
2.0
Goat
IgG2
+++
7.0~8.0
5.8
Mouse
IgG1
++
8.0~9.0
5.5~7.5
IgG2a
+++
7.0~8.0
4.5~5.5
IgG2b
+++
7.0
3.5~4.5
IgG3
+++
7.0
4.0~7.0
Rat
IgG1
+
∶9.0
7.0~8.0
IgG2a
+++
∶9.0
÷8.0
IgG2b
+
∶9.0
÷8.0
IgG2c
++
8.0~9.0
3.0~4.0
Rabbit
IgG
+++
7.4
2.7~4.0
“+” represents the binding strength
Performing a separation:
Binding buffer: 20 mM phosphate buffered saline, pH 7.4
Elution buffer: 100 mM glycine-HCl buffer, pH 2.7
Neutralization buffer: 1M Tris-HCl, pH 9.0
1)         Wash the prepacked 1 ml column with 5~10 column volumes of distilled water to remove 20% ethanol.
2)         Equilibrate the column with 5~10 column volumes of binding buffer.
3)         Dilute 5 ml rabbit serum with binding buffer to 50 ml. Filtrate the diluted serum through a 0.45 レm filter and load the sample.
4)         Wash with 5~10 column volumes of binding buffer.
5)         Elute with 5 column volumes of elution buffer and neutralize collect fractions with neutralization buffer.
6)         After each separation cycle, regenerate the resin by washing with approximately 3~5 column volumes of 0.1 M citrate buffer (pH 2.7).
7)Confirm the purity of the collected antibody by SDS-PAGE analysis (Figure 1, >95% purity).
 
Notice:
1)         It is easy and simple to use the prepacked column. It is not necessary to need any equipment to purify antibody.
2)         Be sure that the antibody is stable under the elution conditions selected. If there is any doubt, titrate the antibody fraction to neutrality immediately upon elution in order not to lose biological activity. To accomplish this, prefill the fraction vessels to 5~10% of the intended fraction volume with a buffer concentrate at about pH7.5 (for example 1 M Tris-HCl or 1 M sodium phosphate).
3)         Make sure temperature of the equilibration buffer identical to that of the packed column to avoid air bubbles. If air has entered the column, the column should be repacked.
4)         It is recommended to regenerate the resin once after 5 times separation cycle in order to extend the service life.
5)         After 10 times separation cycle, wash the resin with 5 column volumes of 20% ethanol and 5~10 column volumes of 70% ethanol sequentially to remove hydrophobic substances.
6)         For longer periods of storage, keep at 4~8ìC in 20% ethanol.
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