PureProteome™ Human Albumin/ Immunoglobulin ... - Millipore

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4 00 3 00 2 00 1 00 20 4 00 3 00 2 00 1 00 20 4 00 3 00 2 00 1 00 20 4 00 3 00 2 00 1 00 20 Albumin and Immunoglobulin Depletion from Serum Samples, continued 6. Add the diluted serum sample to the washed beads. Incubate for 60 minutes at room temperature with continuous mixing or end-over-end rotation. 7. A pulse in a microcentrifuge may be required if liquid has accumulated in the microcentrifuge tube cap. Place the tube back into the magnetic stand. Allow the beads to migrate to the magnet. Remove the depleted serum with a pipette. Transfer to a fresh tube and save. NOTE: This represents the albumin and immunoglobulindepleted serum sample. 8. To maximize recovery of the depleted serum sample, wash the beads 3 times, using 500 μL of 1X PBS for each wash. After each wash, disengage the magnet and vortex vigorously for 10 seconds. Re-engage the magnet and allow the beads to migrate to the magnet. Remove the wash fraction with a pipette and combine with the saved depleted serum. 9. Store the depleted fraction at or below -20 °C for long term storage. NOTE: The sample may be concentrated and/or desalted prior to storage. Using Centrifugation for Concentration or Buffer Exchange (Optional) Amicon® Ultra-2 3K centrifugal filter devices are provided for rapid concentration and buffer exchange/desalting of the sample. Typical processing time is 30–60 minutes to reduce the volume of a depleted sample to 50–150 µL. A physical deadstop in the filter device prevents spinning to dryness and avoids potential sample loss. Concentration and buffer exchange/desalting of the depleted serum sample can be performed in the same device. A detailed user guide for the Amicon® Ultra-2 filter device is provided. 1. Add the pooled depleted serum/wash fraction sample to the Amicon® Ultra-2 filter device. 2. Assemble the filter device and centrifuge per instructions to concentrate the sample to the desired volume. 3. For applications that require buffer exchange or desalting, add the desired buffer and centrifuge again to concentrate the sample. Repeat this wash as required to ensure the desired exchange of buffer. For more detailed instructions, consult the Amicon® Ultra-2 User Guide. Using Centrifugation for Concentration or Buffer Exchange, continued 4. Remove the assembled device from the centrifuge and separate the Amicon® Ultra filter device from the filtrate collection tube. 5. To recover the concentrated solute, invert the Amicon® Ultra filter device and concentrate collection tube. Place in centrifuge and counterbalance with a similar device. Spin for 2 minutes at 1,000 x g to transfer the concentrated sample from the device to the tube. NOTE: For optimal recovery, perform the reverse spin immediately. Alternatively, concentration and buffer exchange may be performed using a different method, such as protein precipitation. Elution of Proteins Bound to PureProteome Albumin/ Immunoglobulin Magnetic Beads (Optional) The bound fraction of proteins may be analyzed by SDS-PAGE to confirm recovery of target protein(s) in the unbound depleted sample. To elute the bound proteins, resuspend the beads 3 times using a minimum of 100 µL of 200 mM Glycine-HCl, pH 2.0–3.0 for each elution. After adding the Glycine-HCl, disengage the magnet and vortex vigorously for 10 seconds. Re-engage the magnet and allow the beads to migrate to the magnet. Remove the eluted fraction with a pipette and save. NOTE: Samples eluted in 200 mM Glycine-HCl, pH 2.0–3.0 may need to be neutralized by adding 1/10 the eluted sample volume of 2 M Tris, pH 8.0 (not included in kit). Alternatively, the bound proteins may be eluted in a 1X SDS- PAGE Reducing Sample Buffer (RSB). To elute the bound proteins, resuspend the beads in a minimum of 100 µL of 1X RSB. After adding the RSB, disengage the magnet and vortex vigorously for 10 seconds. Remove tube and incubate at 70 °C for 10 minutes. Quickly place the tube back in the magnetic stand and allow the beads to migrate to the magnet. Immediately remove the eluted fraction with a pipette and save. Repeat two more times. NOTE: The antibody ligands (~13 MW) may be observed on the gel if the magnetic beads have been incubated in 1X RSB at 70 °C for 10 minutes. Spin to concentrate Spin to concentrate 10K 10K 10K 10K Depleted serum in PBS 50–150 µL of concentrated depleted serum in PBS Add 40 mM Tris exchange buffer Concentrated and bufferexchanged sample 2
Performance Figure 1. Removal of Human Serum Albumin and Immunoglobulin from Serum Figure 2. Human Serum Protein Analysis Pre- and Post-depletion MW 200 150 100 75 50 37 A. Before depletion pH B. After depletion pH Albumin Ig Heavy Ig Light 5.2 6.5 7.5 8.5 5.2 6.5 7.5 8.5 1 2 3 4 5 6 7 220 MW Coomassie blue-stained 2D PAGE gels of total human serum proteins (Panel A) and the fraction depleted of albumin and immunoglobulin (Panel B) using PureProteome Human Albumin/Immunoglobulin Magnetic Beads. 200 µg of total protein was resolved by isoelectric focusing (pH 3.5–10) in the first dimension and 10% SDS-PAGE in the second dimension. 94 60 43 29 14 220 94 60 43 29 14 MW Performance, continued Figure 3. Depletion Efficiency of Human Serum Albumin and Immunoglobulin Subtypes from Human Serum Specifications PureProteome Human Albumin/Immunoglobulin Magnetic Beads Matrix Particle form Bead diameter Storage Mixture of polymer-coated magnetic silica beads covalently coupled with antibody ligands Spherical 10 μm (nominal) 2–8 °C. Do not freeze. % Depletion > 98% albumin and immunoglobulin Typical values are > 99% Shelf life Refer to expiration date on product label PureProteome Human Albumin/Immunoglobulin Magnetic Beads are for research use only. They are not for use in diagnostic procedures. Disposal IgA IgG 1 IgG 2 IgG 3 IgG 4 IgD IgE IgM Albumin Depletion Efficiency 99.79 99.94 99.91 99.98 99.83 99.99 99.11 99.92 99.98 (%) 25 µL human serum was mixed with PureProteome Human Albumin/ Immunoglobulin Magnetic Beads (900 µL of slurry) and depleted as outlined in the protocol. The pre- and post-depleted human serum samples were assayed by ELISA (albumin, IgD, and IgE) or Luminex® assay (IgA, IgG 1, IgG 2, IgG 3, IgG 4 , and IgM) to calculate the percent depletion. Collect and dispose of used material according to all applicable international, federal, state, and local regulations. Safety Data Sheet Safety Data Sheets (SDS) are available on our web site. Go to www.millipore.com and enter your catalogue number in the search box. 25 20 15 10 Lane 1 2 3 4 5 6 7 Sample Sample Volume Molecular Weight Markers 4X Human serum diluted Unbound (depleted fraction) Wash 1 Wash 2 Wash 3 Elution (bound fraction) 10 µL 2 µL 2 µL 2 µL 2 µL 2 µL 2 µL Proteins were resolved on 4–12% SDS-PAGE gel and stained with Coomassie blue. Human serum (25 µL) was depleted of albumin and immunoglobulin following the depletion protocol described. The bound fraction was eluted from the magnetic beads using 100 µL additions of 200 mM Glycine-HCl, pH 2.5. 3
Page 1: LIPO User Guide PureProteome Human

PureProteome™ Human Albumin/ Immunoglobulin ... - Millipore

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