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9969
Rb Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Rb Antibody Sampler Kit #9969

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Rb Antibody Sampler Kit: Image 1

Immunoprecipitation of phospho-Rb (Ser780) from WI-38 cell extracts using Phospho-Rb (Ser780) (D59B7) Rabbit mAb (lane 2). Western blot was performed using the same antibody. Lane 1 is 10% input.

Rb Antibody Sampler Kit: Image 2

Western blot analysis of extracts from human fibroblasts synchronized by serum deprivation, using Phospho-Rb (Ser795) Antibody. Cells were synchronized for 24 hours, then released by addition of serum and harvested at the times indicated. Cell cycle progression was verified by cyclin analysis and FACS. (Provided by John Boylan, Dupont/Merck, Delaware.)

Rb Antibody Sampler Kit: Image 3

Flow cytometric analysis of Jurkat cells using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb and Propidium Iodide (PI/RNase Staining Solution #4087 to measure DNA content. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

Rb Antibody Sampler Kit: Image 4

Chromatin immunoprecipitations were performed with cross-linked chromatin from Raji cells and either Rb (4H1) Mouse 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 using SimpleChIP® Human Timeless Intron 1 Primers #7001, human DHFR promoter primers, 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.

Rb Antibody Sampler Kit: Image 5

Western blot analysis of extracts from HeLa cells (lane 1) or Rb knock-out cells (lane 2) using Rb (4H1) Mouse mAb #9309 (upper), and β-actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the Rb knock-out HeLa cells confirms specificity of the antibody for Rb.

Rb Antibody Sampler Kit: Image 6

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.

Rb Antibody Sampler Kit: Image 7

Western blot analysis of extracts from WI-38 cells, serum-starved for 3 days (-) or serum-starved for 3 days followed by treatment with 10% serum for 2 days (+), using Phospho-Rb (Ser780) (D59B7) Rabbit mAb.

Rb Antibody Sampler Kit: Image 8

Western blot analysis of Rb Control Protein #9303, using Phospho-Rb (Ser795) Antibody (upper) or Rb (4H1) mAb #9309 (lower).

Rb Antibody Sampler Kit: Image 9

Confocal immunofluorescent analysis of MCF7 (left) and BT-549 (right) cells, untreated (upper) or λ phosphatase-treated (lower) using Phospho-Rb (Ser807/Ser811) (D20B12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Rb Antibody Sampler Kit: Image 10

Flow cytometric analysis of Jurkat cells, using Rb (4H1) Mouse mAb versus propidium iodide (DNA content). The box indicates Rb positive cells.

Rb Antibody Sampler Kit: Image 11

Western blot analysis of phosphorylated or nonphosphorylated recombinant, truncated Rb, without or with Rb blocking peptide, using Phospho-Rb (Ser780) (D59B7) Rabbit mAb.

Rb Antibody Sampler Kit: Image 12

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

Rb Antibody Sampler Kit: Image 13

Confocal immunofluorescent image of SH-SY5Y cells, using RB (4H1) Mouse mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

Rb Antibody Sampler Kit: Image 14

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

Rb Antibody Sampler Kit: Image 15

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing nuclear localization, using Rb (4H1) Mouse mAb.

Rb Antibody Sampler Kit: Image 16

Immunohistochemical analysis of paraffin-embedded mouse spleen using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

Rb Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Rb (4H1) Mouse mAb.

Rb Antibody Sampler Kit: Image 18

Immunohistochemical analysis of paraffin-embedded human ovarian serous adenocarcinoma using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Rb Antibody Sampler Kit: Image 19

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-), SignalSilence® Rb siRNA I #6451 (+) or SignalSilence® Rb siRNA II (+), using Rb (4H1) Mouse mAb #9309 and α-Tubulin (11H10) Rabbit mAb #2125. The Rb (4H1) Mouse mAb confirms silencing of Rb expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of Rb siRNA.

Rb Antibody Sampler Kit: Image 20

Immunoprecipitation of phospho-Rb (Ser807/811) from Cos cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

Rb Antibody Sampler Kit: Image 21

Western blot analysis of extracts from COS-7 cells, untreated or hydroxyurea-treated (G1/S), using Rb (4H1) Mouse mAb.

Rb Antibody Sampler Kit: Image 22

Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with calf intestinal phosphatase (CIP) and λ phosphatase (+), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb (upper) or Rb (4H1) Mouse mAb #9309 (lower).

Rb Antibody Sampler Kit: Image 23

Western blot analysis of extracts from WI-38 cells, serum-starved for 3 days (-) or serum-starved for 3 days followed by treatment with 10% serum for 2 days (+), using Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb.

To Purchase # 9969T
Product # Size Price
9969T
1 Kit  (4 x 20 µl) $ 327

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Rb (Ser780) (D59B7) Rabbit mAb 8180 20 µl
  • WB
  • IP
H Mk M R 110 Rabbit IgG
Phospho-Rb (Ser795) Antibody 9301 20 µl
  • WB
  • IP
H Mk R 110 Rabbit 
Phospho-Rb (Ser807/811) (D20B12) XP® Rabbit mAb 8516 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H Mk M R 110 Rabbit IgG
Rb (4H1) Mouse mAb 9309 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
B H Mk Pg 110 Mouse IgG2a
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product Description

The Rb Antibody Sampler Kit provides reagents and protocols to investigate cell cycle progression within cells. The kit contains primary and secondary antibodies to perform two Western blot experiments with each antibody.

Specificity / Sensitivity

All antibodies contained in this kit detect endogenous levels of their respective target protein.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide and are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with recombinant human proteins or synthetic peptides.

Background

The retinoblastoma tumor suppressor protein Rb regulates cell proliferation by controlling progression through the restriction point within the G1-phase of the cell cycle (1). Rb has three functionally distinct binding domains and interacts with critical regulatory proteins including the E2F family of transcription factors, c-Abl tyrosine kinase, and proteins with a conserved LXCXE motif (2-4). Cell cycle-dependent phosphorylation by a CDK inhibits Rb target binding and allows cell cycle progression (5). Rb inactivation and subsequent cell cycle progression likely requires an initial phosphorylation by cyclin D-CDK4/6 followed by cyclin E-CDK2 phosphorylation (6). Specificity of different CDK/cyclin complexes has been observed in vitro (6-8) and cyclin D1 is required for Ser780 phosphorylation in vivo (9).

  1. Sherr, C.J. (1996) Science 274, 1672-7.
  2. Nevins, J.R. (1992) Science 258, 424-9.
  3. Welch, P.J. and Wang, J.Y. (1993) Cell 75, 779-90.
  4. Hu, Q.J. et al. (1990) EMBO J 9, 1147-55.
  5. Knudsen, E.S. and Wang, J.Y. (1997) Mol Cell Biol 17, 5771-83.
  6. Lundberg, A.S. and Weinberg, R.A. (1998) Mol Cell Biol 18, 753-61.
  7. Connell-Crowley, L. et al. (1997) Mol Biol Cell 8, 287-301.
  8. Kitagawa, M. et al. (1996) EMBO J 15, 7060-9.
  9. Geng, Y. et al. (2001) Proc Natl Acad Sci USA 98, 194-9.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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