A part of MilliporeSigma

Montage Antibody Purification Kits

Designed for fast and easy antibody purification from serum, ascites or cell culture supernatants.

Overview

Specifications

Ordering Information

Montage Antibody Purification Kits Clear Sorting & Filtering
Catalog Numbericon Descriptionicon Pack Sizeicon
LSK2ABA20Montage Antibody Purification Kit with PROSEP-A media 2 columns (20 purifications) Show Pricing & Availability
LSK2ABA60Montage Spin Columns with PROSEP-A media 6 columns (60 purifications) Show Pricing & Availability
LSK2ABG20Montage Antibody Purification Kit with PROSEP-G media 2 columns (20 purifications) Show Pricing & Availability
LSK2ABG60Montage Spin Columns with PROSEP-G media 6 columns (60 purifications) Show Pricing & Availability
SCGP00525Steriflip-GP, 0.22 µm, polyethersulfone, gamma irradiated 25 Show Pricing & Availability
UFC903008Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane 8 Show Pricing & Availability
UFC903024Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane 24 Show Pricing & Availability
UFC903096Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane 96 Show Pricing & Availability

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    Documentation

    Data Sheet

    Title
    Montage Antibody Purification Kits

    Posters

    Title
    Poster: Rapid, Ultrafiltration-based Method for Purification of Monoclonal Antibodies from Hybridoma Supernatants

    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

    QuestionAnswer
    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.
    Is pH an important parameter to control during Protein A spin chromotgraphy? The elution pH is the most critical variable. For Protein A, elution by pH steps (starting at pH 6.0) may fractionate different species (weaker binding bovine IgG from target antibodies) or subclasses. High pH (pH 8.0-9.0) in conjunction with high salt may promote binding of mouse IgG to Protein A. The binding buffer pH should normally be higher that pH 6.0-7.0.
    Can I elute antibodies from Prosep A and Prosep G spin columns using divalent cations? Concentrations of divalent cations (particularly Mg2+) up to 1 M can sometimes replace acidic pH if there is concern about loss of activity of acid-labile immunoglobulins.
    There is poor resolution of my target protein after using Prosep A spin columns. What could be the cause of this? There could be 3 causes: The sample volume or concentration may be too large for the capacity of the media plug. Reducing the sample load or volume will fix this. The sample may need to filtered more carefully. The sample is not pure enough.
    What is the binding capacity of the Prosep G spin column? Using the Prosep G spin columns we have been able to bind >10 mg of Rabbitt IgG from serum.
    How can I regenerate the Prosep G spin columns? We recommend that you wash the plugs with 10 ml of Elution Buffer B2 (pH2.5) by centrifuging the spin columns at 500 xg for 5 minutes. Re-equilibrate the plugs with 5 ml of Binding Buffer A by centrifuging the spin columns at 500 xg for 2 minutes. For immediate re-use wash with 5 ml of Binding Buffer A or for later use store the plugs without their end caps in 5 ml of Binding Buffer A in a 15 ml screw-capped tube.
    How do I monitor the purity of the antibodies isolated using the Prosep G spin column? Purity is best monitored by gel electrophoresis. When analyzed by SDS-PAGE under non-reducing conditions IgG antibodies should give a single protein band of about 160-170 kDa. Under reducing conditions (DTT or 2-mercaptoethanol), two or more bands will be seen corresponding to the individual heavy chains (50-55 kDa) or light chains (25 kDA).
    Is the pH an important parameter to control when using the Prosep G spin columns? The elution pH is the most critical variable. The Prosep G spin columns require acidic conditions to desorb the immunoglobulins.
    I am getting poor resolution of my target protein on my gel after eluting from the Prosep G spin column. What could be the cause? There could be 2 causes: The sample volume or concentration may be too large for the capacity of the media plug. reducing the sample load or volume will fix this. The sample may also need to be filtered carefully.
    What is the best way to store the Prosep plugs for a couple of months? The Prosep columns are transported in 20% ethanol. In order to keep the plugs for several months, it is better to take them out from the spin column using the insertion tool and keep them in a container with 20% ethanol. If the customer prefers azide, it will be OK too.
    How do I monitor the purity of isolated antibodies? Purity is best measured by gel electrophoresis. When analyzed by SDS-PAGE under non-reducing conditions, IgG anitbodies should give a single protein band at about 160-170 kDa. On reduction with DTT or 2-mercaptoethanol, two or more bands will be seen corresponding to the individual heavy chains (50-55 kDa) or light chains (25 kDa).
    Are there any critical starting conditions for use of the Prosep A spin columns? For optimal performance, the pH should be above 8.0.
    I am getting poor recovery using the Prosep A and/or Prosep G spin columns with my own buffers. What can I do about this? We strongly recommend that you use the buffers provided with the kit or make up these exact buffers to use with the Prosep A or Prosep G spin columns. This will ensure that you get the best possible recovery.
    The target protein doesn't seem to be eluting from the Prosep A spin columns. What could be the cause of this? There could be 3 causes: The pH of the elution buffer may be incorrect. It would be advisable to prepare a new solution. The elution conditions may be too mild to desorb the target protein. The immunoglobulin was never bound to the Prosep A media.
    Can I reuse the Prosep A spin column with different types of samples? We do not recommend this because of a chance of carry over from the previous sample. Our data however does indicate that after the column has been regenerated it is very clean. Therefore, you are welcome to try it out on your own.
    I see bubbles or cracks in the media bed of the Prosep A and/or Prosep G spin column. What could be the cause of this? The spin column was stored at a cool temperature and then rapidly warmed up. The Prosep A and Prosep G spin columns should be slowly warmed to room temperature before use to prevent this.
    Is there a specific flow rate that I should use with the Prosep A spin columns? Samples (esp. serum & ascites) must be pre-filtered with the Steriflip immediately before applying to the PROSEP device. Your flow rate will depend on the viscosity of your sample. The flow rate to expect with whole serum is about 1 ml/min. If you are seeing a flow rate greater than this you need to decrease the speed of your centrifuge. Having a slower flow rate may help increase the residency time of your sample.
    How should I prepare my sample for the Prosep A and Prosep G spin columns? We recommend pre-filtering using the Steriflip-GP provided in the kit and then diluting 1:1 (v/v) in the Binding Buffer A supplied with the kit.
    Why do you suggest that the Prosep G spin columns be spun at 100-150 xg for 20 minutes during the binding step? The low spin speed for 20 minutes allow for maximum residence time and the best binding.
    Do I need to be careful with the type of chaotropic ion I use when eluting antibodies from the Prosep G spin columns? It is recommended to use the mildest chaotropic aganets at the lowest possible concentration that will ensure rapid elution and high recovery of activity.
    What are the critical starting conditions for the Prosep G spin columns? For optimal performance the pH should be above 8.0.
    Do I need to control the salt concentration when using the Prosep G spin columns? Use 0.1-0.5 M salt to reduce non-specific adsorption.
    The sample does not flow easily through the Prosep A and Prosep G spin column. what could be the cause? There could be 3 causes:

    1. The media is clogges with particulates, most likely because the sample was not pre-filtered, or because the serum is particularly thick.
    2. If the spin columns are not stored at 2-8 C microbial growth could occur in the media plug causing a restriction of flow.
    3. The g force for the sample loading step should be increased up to 150 x g.
    The target protein doesn't seem to be eluting from the Prosep G spin column. Could there be a cause? The pH of the elution buffer may be inccorect. If it is not acidic enough, the target protein will not desorb from the spin column. It would be advisible to prepare a new solution and check the pH.
    The recovery of my target protein from the Prosep Aand Prosep G spin columns is low, what could be the cause? The binding of antibodies to the Prosep A and Prosep G spin columns is attributed in part to hydrophobic forces. Chaotropic salts can be added to the elution solution reducing the strength of the hydrophobic interactions.
    My target Protein eluted from the Prosep G spin column is running at an unexpected position on the gel. What could be the cause? There could be 2 causes: There may be an ionic interaction between the protein and the media in the Prosep G spin column. You should maintain the ionic strength above 50mM. There may ne hydrophobic interactions between the sample and the media in the Prosep G spin column. In thisinstance reduce the salt concentration ans add suitable detergent or solvents.
    My Buffer A Solution from my Montage Antibody Purification Kit Prosep A (or Prosep G) has turned slightly yellow. I know the shelf life of the kit is 2 years but is this solution ok to use? Yes, this slight change in color will not affect the activity of the Buffer A Solution.
    Can the antibody purification with the Montage Antibody Purification Prosep A/G columns be done with gravimetric flow instead of centrifugation? No. There is not enough force to run via gravimetric flow. These devices designed for centrifugation.

    User Guides

    Title
    Montage Antibody Purification Kit and Spin Columns with PROSEP-A Media
    Montage Antibody Purification and Spin Columns with PROSEP-G Media

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    Categories

    Life Science Research > Protein Detection and Quantification > Immunoassays > Immunoprecipitation (IP) > Agarose Beads for IP & Antibody Purification
    Life Science Research > Antibodies and Assays > Immunoassays > Immunoprecipitation (IP) > Agarose Beads for IP & Antibody Purification
    Life Science Research > Protein Sample Preparation > Protein Purification > Agarose Bead Affinity Purification > Agarose Beads for IP & Antibody Purification