Montage Antibody Purification Kits
Designed for fast and easy antibody purification from serum, ascites or cell culture supernatants.
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Designed for fast and easy antibody purification from serum, ascites or cell culture supernatants. Less
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Overview
Specifications
Ordering Information
Documentation
References
| Reference overview |
|---|
| Antibody Purification with Affinity Spin Columns Kavonian, Mark,BioScience Technology, July 2003 BioScience Technology, July 2003 2003 |
| Considerations During Development of a Protein A-Based Antibody Purification Process Iyer et al.,Biopharm, January 2002, pp 14-20 Biopharm, January 2002, pp 14-20 2002 |
| A sensitive enzyme linked immunoadsorbent assay (ELISA) for the detection of staphylococcal prtein A (SpA) present as a trace contaminant of murine immunoglobulins purified on immobilized protein A Miguel A.J. Godfrey, Piotr Kwasowski, Roland Clift and Vincent Marks Journal of Immunological Methods, 149(1992) 21-27, 1992 1992 |
FAQ
| Question | Answer |
|---|---|
| What is the shelf life of the Prosep A and Prosep G spin columns? | The spin columns/media are under warranty for 2 years provided they are stored at 2-8 C. |
| Do I need to filter the buffers provided with the Montage Antibody Purification kit? | The buffers supplied with the kit are pre-filtered for immediate use. |
| Do I need to pre-filter my sample before loading it onto a Prosep A or ProSep G spin column? | All samples must be filtered through a 0.22 mm filter immediately before loading the sample onto the column. We recommend using a Steriflip-GP 0.22 mm filter. |
| What is the binding capacity of the Prosep A spin column? | Using the Prosep A spin columns we have been able to bind >20 mg of Rabbit IgG. |
| What is the maximum volume I can load onto the Prosep A and Prosep G spin columns? | You can load up to 20 ml in a Prosep A or Prosep G sping column for a swinging bucket rotor and up to 10 ml for a fixed angle rotor. We recommend that you spin it in a swinging bucket rotor. |
| Why do you suggest that the Prosep A spin columns be spun at 100-150 xg for 20 minutes? | The low speed, twenty minute centrifugation maximizes IgG / resin contact which increases IgG binding efficiency. |
| Can I use a fixed angle rotor or a swinging bucket rotor with the Prosep A and Prosep G spin columns? | Swing bucket rotors are recommended because they provide uniform flow through the resin. For optimal performance with a fixed angle rotor, ensure that the orientation of the Prosep A spin coulmn is the same for all steps. Also please note that the maximum sample loading volume for a fixed angle rotor is ten ml versus twenty ml with a swinging bucket rotor. |
| What is the minimum elution volume for the Prosep A and Prosep G spin columns? | We suggest 10 ml. Smaller volumes can be used but they may result in incomplete IgG recovery. If you use a smaller volume the neutralization buffer volume should also be adjusted accordingly. |
| How do I regenerate the Prosep A spin columns? | We recommend that you wash the plugs with 10 ml of Elution Buffer B2 by centrifuging at 500 xg got 5 minutes. re-equilibrate the media with 5 ml of Binding Buffer A by centrifuging the spin column at 500 xg for 2 minutes. Proceed to step 1 of the antibody purification protocol for immediate re-use or store the plugs without their end caps in 5 ml of Binding Buffer a in a 15 ml screw-capped tube for future use. |
| Can I autoclave the Prosep A and Prosep G spin columns? | The Prosep A and Prosep G spin columns cannot be autoclaved. |
Related Products & Applications
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Categories
Montage Antibody Purification Kits are designed for fast and easy antibody purification from serum, ascites or cell culture supernatants. The kits include spin columns pre-packed with high capacity PROSEP-A or G media. There are no pumps to setup. No columns to pack. All you need is a centrifuge to purify 10–20 mg in less than 60 minutes.
The protocol is easy to follow and, unlike traditional gravity flow methods, the use of centrifugal purification allows for walk-away convenience.
Results show that the spin columns can be regenerated up to 10X with no loss of binding capacity or reduced
specificity.
Performance
Spin Column Reuse
)
Human IgG was purified 10 times from normal serum using a regenerated PROSEP-G spin column.
Purification of MAbs
)
Monoclonal mouse IgG was purified with a PROSEP-A spin column from ascites. (Lane 1: unpurified ascites. Lane 2: flow-through ascites. Lane 3: wash flow-through. Lane 4: 5 µg purified antibody.) Courtesy Exalpha Biologicals.
| Binding Affinities of Protein A and Protein G | ||||||
| Protein | A | G | Protein | A | G | |
| Human IgG1 | ++ | ++ | Rat IgM | + /-- | - | |
| Human IgG2 | ++ | ++ | Rabbit IgG | ++ | ++ | |
| Human IgG3 | -- | ++ | Hamster IgG | + | ++ | |
| Human IgG4 | ++ | ++ | Guinea Pig IgG | ++ | + | |
| Human IgA | + | -- | Bovine IgG | + | + | |
| Human IgD | + | -- | Sheep IgG | + / -- | + | |
| Human IgE | + | -- | Goat IgG | + /-- | + | |
| Human IgM | + | -- | Pig IgG | ++ | ++ | |
| Mouse IgG1 | + | + | Chicken IgG | -- | + /-- | |
| Mouse IgG2a | ++ | ++ | Fragments | |||
| Mouse IgG2b | ++ | ++ | Human Fab | + | + | |
| Mouse IgG3 | + | ++ | Human F(ab')2 | + | + /-- | |
| Mouse IgM | + /-- | -- | Human ScFv | + | -- | |
| Rat IgG | ++ | ++ | Human Fc | + | -- | |
| Rat IgG1 | + /-- | + | Human K | -- | -- | |
| Rat IgG2a | + /-- | ++ | Human λ | -- | + | |
| Rat IgG2b | + /-- | + | ||||
| Rat IgG2c | + /-- | + | ||||
| Legend: ++ = Strong affinity; + = Moderate/slight affinity; +/-- = Requires evaluation; -- = No affinity | ||||||
| Kit Contents | Includes spin columns, buffers, sample preparation and concentration devices | |
| Supporting Matrix Material | Porous glass | |
| Media Particle Size, µm | 75 µm–125 µm | |
| Media Bed Volume, µL | 1.6 mL | |
| Binding Capacity | ||
| Protein A | >20 mg of rabbit IgG | |
| Protein G | >10 mg of rabbit IgG | |
| pH Range | pH2 – pH9 | |
| Maximum Volume, mL | 20 mL (maximum in swinging bucket rotor) | |
| Storage Temperature | 2 to 8 °C for media. Buffer at room temperature. | |
| Color Coded End-caps | ||
| Protein A | Red | |
| Protein G | Yellow | |