Cat. No. MG17PG-1ml XPRESSAFFINITY PROTEIN G- MAGNETIC NANOPARTICLES (MNP) FOR RESEARCH APPLICATIONS Product Description: MagGenome s Protein G-MNP provides a fast and convenient method for affinity based isolation of proteins using the magnetic method. Protein G is immobilized on magnetic nanoparticles (size range: 10-20 nm) using our novel patented technology. The blocking step during preparation of the resin minimizes non-specific binding of other contaminating proteins present in the biological samples and also minimizes leaching of the immobilized protein from the matrix. The product is provided as 10 mg magnetic particles in 1 ml of phosphate buffer ph 8, containing 0.05% Tween-20 and 0.05% sodium azide. Recommended storage temperature is 4 0 C. Advantages: The advantages of the product include high binding capacity due to large surface area of nanoparticles and high magnetic properties due to the lack of polymeric coating material on the particles. Since this is a magnetic separation system, no centrifugation or packing of columns is involved. Unlike Sepharose beads, washing of magnetic beads is quick and convenient and there is absolutely no loss of particles during processing. Applications: Protein G is generally used for the isolation of antibodies from serum, cell culture supernatant, ascites etc. and for immunoprecipitation and co-immunoprecipitation of antigens from cell or tissue extracts. For antibody purification, the beads are incubated with the antibody solution and then magnetically separated from the supernatant. For immunoprecipitation, the beads are added to an antigen-containing sample to which antibody has been added and allowed to incubate to form the antibody-antigen complex. This product can be used for chromatin immunoprecipitation (ChIP) and RNA-IP as well. Protein G can bind to antibodies from many Page no - 1
species like human, mouse and rat. Currently the product is recommended only for research applications and not for any therapeutic purpose. Magnetic bead based products are highly favoured by research labs over Sepharose for small scale applications such as IP, ChIP and RNA-IP but can also be scaled up for bulk applications. Performance of the product: The product performed consistently during stability tests and is fully active till12 months from the date of preparation. Though the recommended storage is 4 0 C, the product is also stable at room temperature for short term. Binding capacity of Protein G-MNP is approximately 120-150 g human IgG per mg of MNPs which is at par or even higher than other magnetic beads available in the market. The product should not undergo freezing or vortexing and should be dispersed by gentle pipetting. PROTOCOLS 1. ANTIBODY PURIFICATION FROM WHOLE BLOOD, SERUM, ASCITES, CELL CULTURE MEDIUM OR CELL LYSATE (Small scale purification of IgG/Fab using XpressAffinity Protein G-Magnetic Nanoparticles) (1 ml product per vial: supplied at 10 mg magnetic particles/ml in 5mM Phosphate buffer ph 8 containing 0.05% Tween-20 and 0.05% Sodium Azide) Storage: Upon receipt store at 4 C. Note: Do not centrifuge, dry or freeze the product. Mix the beads thoroughly before use by repeated inversion or gentle pipetting up and down (Do not vortex). Protocol: 1. Place 100μl of Protein G magnetic nanoparticles into a 1.5 ml micro-centrifuge tube. Add 900μl of 1X PBS and gently mix. 2. Place the tube on a magnetic stand to collect the particles against the side of the tube. Discard the supernatant. 3. Add 1ml of 1X PBS again and invert/tap the tube several times to mix. Collect particles with magnetic stand and discard the supernatant. Repeat wash twice. 4. Re-suspend the particles in 100μl of 1X PBS. Page no - 2
5. Add 500μl sample (serum, plasma or ascites) to this tube and invert to mix. Make up to 1ml with 400μl of PBS. 6. Please note: For purification of secreted antibodies in cell culture, we recommend using 1ml of Protein G nanoparticles for 200 ml culture supernatant. If you are purifying antibodies from cell lysate, use 1ml nanoparticles per 20-25ml lysate. 7. Incubate the samples at room temperature with mixing for 2 hours using a tube rotator. 8. Please Note: If you are performing large scale purifications as with cell culture medium or lysate you may incubate overnight at 4 0 C. 9. After incubation, collect the particles on a magnetic stand and discard the supernatant. 10. Add 1ml of PBS to the tube, mix well, collect the beads with a magnetic stand and discard the supernatant. Repeat this wash thrice. 11. Add 300μl of Elution Buffer (preferably use 0.2 M L-Arginine ph 3.0) to the tube, mix well and incubate 5 minutes at room temperature with gentle mixing. 12. Collect the particles with a magnetic stand and then remove and save the supernatant that contains the eluted antibody (E1). To neutralize the low ph, add the required amount of neutralization buffer (1M Tris, ph 9) so that ph becomes 7. 13. Measure the concentration of IgG on a Nanodrop or by a standard protein assay (like Bradford s assay). 14. Repeat the elution step and collect the eluted antibody as E2, E3 etc until the IgG gets completely eluted (each elution for 5 minutes). Neutralize all eluates and measure concentration. 15. Analyze the samples by running on an SDS-PAGE. 2. IMMUNOPRECIPITATION USING XPRESSAFFINITY PROTEIN G-MAGNETIC NANOPARTICLES Important product information 1. Do not centrifuge, dry or freeze the product. 2. To minimize protein degradation, include protease inhibitors in the preparation of cell lysate. Protocol for Immunoprecipitation (This is only a guideline for immunoprecipitation and will require optimization for different types of analyses) 1. Incubation of lysate with antibody: Prepare the cell lysate and incubate with the required amount of primary antibody against the target antigen following your lab protocol. (Incubate at 4 0 C from a few hours to overnight). Page no - 3
2. Take out the magnetic nanoparticles from 4 0 C storage and resuspend thoroughly by gentle tapping or by pipetting up and down. Take 50μl of Protein G- Magnetic Nanoparticles in a 1.5 ml tube. 3. Wash: Add 450μl of Lysis buffer (or 1X PBS) to the beads and gently mix by inverting the tube (make sure that particles are uniformly re-suspended in solution-do not vortex). 4. Place the tube on a magnetic stand to collect the nanoparticles against the side of the tube. Remove the supernatant. 5. Remove the tube from the stand. Add 1ml of Lysis buffer (or PBS) to the tube. Invert the tube several times to mix. Collect the particles with magnetic stand. Remove the supernatant. 6. Repeat the washing step. Remove supernatant completely. 7. Incubation of nanoparticles with antibody-antigen complex: Remove the tube from the stand. Add the lysate-antibody mixture to the tube which contains the nanoparticles, gently mix to resuspend completely (do not vortex) and incubate at 4 0 C for 2 hours with mixing (preferably continuous mixing on a tube rotator). (If you prefer to give an overnight incubation, keep at 4 0 C with continuous rotation). 8. Collect the nanoparticles with a magnetic stand and then remove the flow-through (save for SDS-PAGE if required). 9. Remove tube from the stand. Add 500μl of PBS to the tube and gently mix. Collect the particles on magnetic stand and discard the supernatant. Repeat wash three times. 10. Elution: 11. Add 50-100μl of 1X SDS-PAGE reducing sample buffer to the nanoparticles and re-suspend and heat samples at 96-100ºC in a heating block for 10 minutes. Magnetically separate the beads and collect the supernatant containing the target antigen. Proceed with SDS-PAGE and Western blotting. TROUBLESHOOTING GUIDE Problem Possible reasons Suggestions/solutions Protein was degraded in the source material Add protease inhibitors during the preparation of lysate Page no - 4
Low protein recovery Eluted protein was found inactive Magnetic particles were found to be aggregated The amount of magnetic particles used may be low The amount of target protein may be low in the sample Elution conditions were mild Elution condition was too stringent Particles were not properly stored or treated Optimize the amount of particles needed Increase the amount of target protein Increase the incubation time with the elution buffer Use elution buffer with ph3.0 Neutralize immediately after elution Do not freeze or vortex Pipet gently up and down or tap mix IgG band too intense on blot Too much beads were taken Optimize the amount of beads required for your experiment FREQUENTLY ASKED QUESTIONS 1. How do we choose between Protein A and Protein G? Protein A and Protein G can bind to IgGs, but their binding properties differ among species and subclasses of IgG. -Protein A is preferred for human, rabbit, pig, dog, and cat IgG. -Protein G has better binding capacity for a broader range of mouse, rat and human IgG subclasses. 2. What is the maximum binding capacity of Protein G-magnetic nanoparticles? The maximum binding capacity of Protein G-magnetic nanoparticles is approximately 120-150 g IgG per 1 mg of the nanoparticles. 3. In what pack sizes the product is available? Protein G-magnetic nanoparticles are currently available as 1ml and 5ml products. 4. What are the advantages of the product over existing products/technologies? MagGenome s Protein G-Magnetic nanoparticles have significantly higher binding capacity compared to most of the Protein G-magnetic beads available in the market. The protocol is Page no - 5
quick and simple because all washing and elution steps are performed with the help of an external magnetic field. It is ensured that the loss of the affinity resin is nil during the process compared to Sepharose beads. 5. How is the stability of the product? The recommended storage condition of the product is 4 0 C. The product is stable for one year under ideal storage conditions. 6. What are the applications tested for the product? Small scale antibody purification, Immunoprecipitation, Chromatin immunoprecipitation, RNA-IP Page no - 6