The µMACS and MultiMACS Protein A/G Kits facilitate rapid analytical scale immunoprecipitation (IP)
2
, Co-IP
1
, or chromatin immunoprecipitation (ChIP)
8,10,20
of proteins and their interaction partners from cell lysates. They can be used in combination with antibodies or serum specific for the target protein of choice.
The µMACS and MultiMACS Protein A/G Kits contain colloidal super-paramagnetic MicroBeads, which are conjugated to Protein A/G, respectively. The non-sedimenting Protein A/G MicroBeads ensure rapid reaction kinetics enabling the formation of the labeled immune complex in only 30

Data and images for Protein A or Protein G Kits

Figures

Figure 1

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Immunoprecipitation using µMACS Protein A or Protein G MicroBeads.

Figure 1

Immunoprecipitation using µMACS Protein A or Protein G MicroBeads.

Figure 2

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Immunoprecipitation of SV40 large T antigen. SV40 large T antigen was immunoprecipitated from COS cell lysates using a SV40 large T antigen specific antibody and µMACS Protein G MicroBeads. The cell lysate and immunoprecipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis, which was subsequently silver stained.
L: cell lysate, M: protein marker, lane 1 (indicated by arrow): immunoprecipitated SV40 large T antigen, lane 2: IP with isotype-matched control antibody, lane 3: control IP without antibody.

Figure 2

Immunoprecipitation of SV40 large T antigen. SV40 large T antigen was immunoprecipitated from COS cell lysates using a SV40 large T antigen specific antibody and µMACS Protein G MicroBeads. The cell lysate and immunoprecipitated proteins were subjected to SDS-polyacrylamide gel electrophoresis, which was subsequently silver stained.
L: cell lysate, M: protein marker, lane 1 (indicated by arrow): immunoprecipitated SV40 large T antigen, lane 2: IP with isotype-matched control antibody, lane 3: control IP without antibody.

Figure 3

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Co-immunoprecipitation of β-catenin. Androgen receptor was immunoprecipitated from cell lysates of dihydrotestosterone (DHT)-stimulated (lane 1, 3) or unstimulated LNCaP cells (2, 4) using an androgen receptor specific antibody and µMACS Protein G MicroBeads (1, 2) or Protein G agarose beads (lanes 3, 4). Precipitated proteins were analyzed by SDS-PAGE and Western blot using an anti-β-catenin antibody.
Higher sensitivity was achieved when the Co-IP was performed using µMACS Protein G MicroBeads. (Courtesy of D. Mulholland, Vancouver, Canada)

Figure 3

Co-immunoprecipitation of β-catenin. Androgen receptor was immunoprecipitated from cell lysates of dihydrotestosterone (DHT)-stimulated (lane 1, 3) or unstimulated LNCaP cells (2, 4) using an androgen receptor specific antibody and µMACS Protein G MicroBeads (1, 2) or Protein G agarose beads (lanes 3, 4). Precipitated proteins were analyzed by SDS-PAGE and Western blot using an anti-β-catenin antibody.
Higher sensitivity was achieved when the Co-IP was performed using µMACS Protein G MicroBeads. (Courtesy of D. Mulholland, Vancouver, Canada)

Figure 4

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ChIP of androgen receptor. LNCaP prostate cancer cells were stimulated with dihydrotestosterone (DHT) (lanes 1, 3) or vehicle (lanes 2, 4) for 48 h. Subsequently, ChIP was carried out using an androgen receptor specific antibody and µMACS Protein G MicroBeads (lanes 1, 2) or Protein G agarose beads (lanes 3, 4).
ChIP experiments carried out with µMACS Protein G MicroBeads showed enhanced immunoprecipitation and a significantly reduced level of non-specific PCR product. (Courtesy of D. Mulholland, Vancouver, Canada)

Figure 4

ChIP of androgen receptor. LNCaP prostate cancer cells were stimulated with dihydrotestosterone (DHT) (lanes 1, 3) or vehicle (lanes 2, 4) for 48 h. Subsequently, ChIP was carried out using an androgen receptor specific antibody and µMACS Protein G MicroBeads (lanes 1, 2) or Protein G agarose beads (lanes 3, 4).
ChIP experiments carried out with µMACS Protein G MicroBeads showed enhanced immunoprecipitation and a significantly reduced level of non-specific PCR product. (Courtesy of D. Mulholland, Vancouver, Canada)

Specifications for Protein A or Protein G Kits

Overview

The µMACS and MultiMACS Protein A/G Kits facilitate rapid analytical scale immunoprecipitation (IP)
2
, Co-IP
1
, or chromatin immunoprecipitation (ChIP)
8,10,20
of proteins and their interaction partners from cell lysates. They can be used in combination with antibodies or serum specific for the target protein of choice.
The µMACS and MultiMACS Protein A/G Kits contain colloidal super-paramagnetic MicroBeads, which are conjugated to Protein A/G, respectively. The non-sedimenting Protein A/G MicroBeads ensure rapid reaction kinetics enabling the formation of the labeled immune complex in only 30 minutes. Compared to standard protocols, on-column IPs result in reduced non-specific background binding and less protein dissociation from the complex. Moreover, laborious centrifugation or buffer removal steps can be avoided.

Detailed product information

Detailed procedure

After cell lysis the lysate is incubated with the specific antibody or serum against the target protein. Pre-clearing of the lysate is dispensable. Then the antibody-protein complex is magnetically labeled with µMACS Protein A/G MicroBeads. The sample is loaded onto a MACS Column placed in the magnetic field of the µMACS or thermoMACS Separator. After washing, the magnetically labeled antibody-protein complex and associated molecules are retained on the column. After elution the (co-)immunoprecipitated proteins can be analyzed by SDS-PAGE and Western blot.

Applications

The advantages of µMACS Protein A/G MicroBeads for ChIP are shown in a customer protocol and have been incorporated into a special protocol. In contrast to the standard ChIP protocol, this streamlined protocol saves 90% of laboratory time in particular on pre-clearing, incubation times, and reverse crosslinking steps. In addition, it allows for working with limited starting materials of just 10
6
cells. Moreover, customer data indicated enhanced ChIP and a significantly reduced level of non-specific PCR products.

Downstream applications

The (co-)immunoprecipitated proteins can be analyzed by SDS-polyacrylamide gel electrophoresis and subsequent Western blotting techniques
2-3,5-6,9,13
or by on-column enzymatic reactions using the thermoMACS Separator.

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Protein A or Protein G Kits

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