Less than 2 hours—with the unique MACS® Technology, immunopurification takes just 1–2 hours, while conventional immunoprecipitations can last up to one day.
Minimal background—the minimized non-specific binding of MACS MicroBeads and the efficient washing in the column yield highly pure protein.
Convenient—centrifugation or resuspension steps are not required for immunopurification on MACS Columns.
Gentle and versatile—MACS Column Technology gives high yield of target protein, interacting partners, or molecular complexes, for example when used for chromatin-IP (ChIP) or ribosome display.
The easy way of analytical small-scale immunoprecipitation (IP). The extremely small (diameter 50 nm) µMACS™ Protein A or µMACS™ Protein G MicroBeads ensure very rapid reaction kinetics, so that the formation of the labeled immune complex is generally completed in 30 minutes: The target protein is bound by a specific antibody, while µMACS Protein A or µMACS Protein G MicroBeads bind to the Fc region of the specific antibody; thereby, magnetically labeling the immune complex.
As non-specific protein binding of MACS MicroBeads is negligible compared to sepharose or agarose carrier material, time-consuming and yield-reducing pre-absorption steps of conventional IP are not necessary. Next, the immune complex is immobilized in a µ Column placed in the magnetic field of a µMACS or thermoMACS™ Separator.
MACS Column Technology now allows a very gentle but stringent washing of the target protein simply by adding buffers. This convenient procedure is especially advantageous when handling hazardous material such as radioactively labeled proteins.
Sensitive downstream analyses. Finally, the immunopurified protein is eluted in a small volume, while the MicroBeads remain in the column. By eluting with protein sample buffer, the target protein can directly be loaded on an SDS-PAGE gel for protein staining or Western blotting1, 2.
Alternatively, an enzymatic reaction with the immunoprecipitated protein, such as a kinase assay, can be performed in the µ Column. The thermoMACS Separator provides incubation temperatures of 37 °C and 42 °C in the µ Column.
µMACS Protein A and G MicroBeads not only improve standard IP but significantly accelerate the search for interacting proteins3,4. This includes their use for chromatin immunoprecipitation (ChIP)5 or ribosome display6.
The immunopurification can be upscaled 3–5 times by using M Columns in combination with MiniMACS™ or OctoMACS™ Separator. |
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| Figure 1 |
| Working scheme for immunoprecipitation using μMACS™ Protein A or Protein G MicroBeads. |
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| Figure 2 |
| Immunoprecipitation of the SV40 large T antigen from COS cells using μMACS Protein G MicroBeads. Silver stained SDS gel of the cell lysate (L), a protein marker (M), the immunoprecipitated large T antigen (lane 1, indicated by the arrow), an isotype-matched control antibody (2, immunoprecipitation with rat anti-mouse antibody), and a control without antibody (3) are shown. |
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| Figure 3 |
| Detection of biotinylated surface antigens of the B cell line JOK-I following immunoprecipitation. For immunoprecipitation anti-CD22 (HD239, lane 2) or anti-MHC class I (W6/32, 4) antibodies were used. An isotype-matched antibody (1, 3) was used as a control. Following SDS-PAGE and transfer to a membrane, biotinylated proteins were detected using streptavidin-conjugated peroxidase. (Courtesy of Dr. Reinhard Schwartz-Albiez, Heidelberg, Germany.) |
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| Figure 4 |
| Co-immunoprecipitation of Beta-Catenin (BCat): Androgen Receptor was immunoprecipitated from Dihydrotestosterone (DHT)-stimulated (lane 1, 3) or unstimulated LNCaP cells (2, 4) with µMACS Protein G MicroBeads (1, 2) or with Protein A/G agarose beads (lanes 3, 4 ). Western blot (WB) using anti-Beta-Catenin antibody shows Beta-Catenin co-immunoprecipitated with Androgen Receptor. (Courtesy of D. Mulholland, Vancouver, Canada) |
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