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43698
Mediator Complex Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Mediator Complex Antibody Sampler Kit #43698

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Mediator Complex Antibody Sampler Kit: Image 1

Immunoprecipitation of MED1 from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is MED1 Antibody. Western blot analysis was performed using MED1 Antibody.

Mediator Complex Antibody Sampler Kit: Image 2

Immunoprecipitation of MED12 from Jurkat cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or MED12 (D9K5J) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using MED12 (D9K5J) Rabbit mAb.

Mediator Complex Antibody Sampler Kit: Image 3

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells heat shocked for 1 hr and either MED26 (D4B1X) Rabbit mAb or of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human c-Myc Intron 1 Primers #14905, SimpleChIP® Human HSP70 Intron 1 Primers #14023, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Mediator Complex Antibody Sampler Kit: Image 4

Immunoprecipitation of CDK8 from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb XP® Isotype Control #3900, and lane 3 is CDK8 (D6M3J) Rabbit mAb. Western blot analysis was performed using CDK8 (D6M3J) Rabbit mAb.

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.

Mediator Complex Antibody Sampler Kit: Image 5

Flow cytometric analysis of HCT 116 cells, either wild-type (green) or cyclin C knockdown (blue), using Cyclin C (E6V4Z) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Mediator Complex Antibody Sampler Kit: Image 6

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and Rpb1 NTD (D8L4Y) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across the ZNF740 gene on chromosome 12. For additional ChIP-seq tracks, please download the product data sheet.

Mediator Complex Antibody Sampler Kit: Image 7

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

Mediator Complex Antibody Sampler Kit: Image 8

Western blot analysis of extracts from various cell lines using MED1 Antibody.

Mediator Complex Antibody Sampler Kit: Image 9

Western blot analysis of extracts from various cell lines using MED12 (D9K5J) Rabbit mAb.

Mediator Complex Antibody Sampler Kit: Image 10

Western blot analysis of extracts from Raji, HeLa, and Daudi cells using MED26 (D4B1X) Rabbit mAb.

Mediator Complex Antibody Sampler Kit: Image 11

Western blot analysis of extracts from HeLa, Raw 264.7, and COS-7 cell lines using CDK8 (D6M3J) Rabbit mAb.

Mediator Complex Antibody Sampler Kit: Image 12

Immunoprecipitation of cyclin C protein from HCT 116 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cyclin C (E6V4Z) Rabbit mAb. Western blot analysis was performed using Cyclin C (E6V4Z) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody. 

Mediator Complex Antibody Sampler Kit: Image 13

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hela cells and Rpb1 NTD (D8L4Y) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 7 (upper), including ACTB (medium), a known target gene of Rpb1-NTD (see additional figure containing ChIP-qPCR data), and ZNF740 gene on chromosome 12 (lower).

Mediator Complex Antibody Sampler Kit: Image 14

Western blot analysis of extracts from various cell lines using Cyclin C (E6V4Z) Rabbit mAb.

Mediator Complex Antibody Sampler Kit: Image 15

Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells and either Rpb1 NTD (D8L4Y) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human γ-Actin Promoter Primers #5037, SimpleChIP® Human AFM1 Intron 1 Primers #5098, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

Mediator Complex Antibody Sampler Kit: Image 16

Western blot analysis of extracts from control HCT 116 cells (lane 1) or cyclin C knockdown HCT 116 cells (lane 2) using Cyclin C (E6V4Z) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The reduction in signal in the cyclin C knockdown HCT 116 cells confirms specificity of the antibody for cyclin C.

Mediator Complex Antibody Sampler Kit: Image 17

Western blot analysis of extracts from HeLa, KNRK, and COS-7 cells using Rpb1 NTD (D8L4Y) Rabbit mAb.

To Purchase # 43698T
Product # Size Price
43698T
1 Kit  (6 x 20 µl) $ 458

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
MED1 Antibody 51613 20 µl
  • WB
  • IP
H M R Mk 220 Rabbit 
MED12 (D9K5J) Rabbit mAb 14360 20 µl
  • WB
  • IP
H M R Mk 240 Rabbit IgG
MED26 (D4B1X) Rabbit mAb 14950 20 µl
  • WB
  • IP
  • ChIP
H 70 Rabbit IgG
CDK8 (D6M3J) Rabbit mAb 17395 20 µl
  • WB
  • IP
H M Mk 53 Rabbit IgG
Cyclin C (E6V4Z) Rabbit mAb 68179 20 µl
  • WB
  • IP
  • F
H M 30 Rabbit IgG
Rpb1 NTD (D8L4Y) Rabbit mAb 14958 20 µl
  • WB
  • ChIP
H M R Mk 250 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Mediator Complex Antibody Sampler Kit provides an economical means of evaluating total levels of Mediator Complex proteins. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the Mediator Complex Antibody Sampler Kit detects endogenous levels of its target protein.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Gly1813 of human MED12 protein, Pro120 of human MED26 protein, Pro455 of human CDK8 protein, Pro263 of human cyclin C protein, and Glu613 of human Rpb1 protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His591 of human MED1 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

The mammalian Mediator Complex is a multi-subunit protein complex that acts to relay signals from transcription factors to RNA polymerase II (Pol II) and the basal transcription machinery. In other words, Mediator acts as a centralized hub or integrator for transcriptional regulation (reviewed in 1,2). Multiple studies have shown that Mediator functions include epigenetic regulation, transcriptional elongation, transcription termination, mRNA processing, noncoding RNA activation, and super enhancer formation. Through these functions, Mediator contributes to the regulation of many biological processes, including insulin signaling (3), NF-κB-dependent signaling (4), stem cell pluripotency and self renewal (5,6), and proliferation of colon cancer cells (7,8). The Mediator Complex consists of 31 protein subunits and can be divided into four distinct modules: the head, middle, tail, and CDK8 kinase module. The CDK8 kinase module contains the CDK8, cyclin C, MED12, and MED13 subunits and is thought to act as a molecular switch, phosphorylating the C-terminal domain (CTD) of Rpb1, the large subunit of RNA polymerase II, to inhibit Pol II recruitment and transcription initiation (9,10).

  1. Malik, S. and Roeder, R.G. (2005) Trends Biochem Sci 30, 256-63.
  2. Soutourina, J. (2018) Nat Rev Mol Cell Biol 19, 262-274.
  3. Wang, W. et al. (2009) Dev Cell 16, 764-71.
  4. van Essen, D. et al. (2009) PLoS Biol 7, e73.
  5. Tutter, A.V. et al. (2009) J Biol Chem 284, 3709-18.
  6. Varelas, X. et al. (2008) Nat Cell Biol 10, 837-48.
  7. Firestein, R. et al. (2008) Nature 455, 547-51.
  8. Morris, E.J. et al. (2008) Nature 455, 552-6.
  9. Knuesel, M.T. et al. (2009) Mol Cell Biol 29, 650-61.
  10. Knuesel, M.T. et al. (2009) Genes Dev 23, 439-51.

Pathways & Proteins

Explore pathways + proteins related to this product.

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