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2562
p190-B RhoGAP Antibody
Primary Antibodies
Polyclonal Antibody

p190-B RhoGAP Antibody #2562

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  1. WB
Western Blotting Image 1 - p190-B RhoGAP Antibody

Western blot anlaysis of extracts from MCF-7 (human), C2C12 (mouse), and BAEC (bovine) cells, using p190-B RhoGAP Antibody.

To Purchase # 2562S
Product # Size Price
2562S
100 µl $ 268

Supporting Data

REACTIVITY H M R Mk B
SENSITIVITY Endogenous
MW (kDa) 190
SOURCE Rabbit

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 10

Specificity / Sensitivity

p190-B RhoGAP Antibody detects endogneous levels of total RhoGAP protein.

Species Reactivity:

Human, Mouse, Rat, Monkey, Bovine

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a region surrounding Lys296 of human p190-B RhoGAP. Antibodies are purified by protein A and peptide affinity chromatography.

Background

Rho family GTPases are key regulators of diverse processes such as cytoskeletal organization, cell growth and differentiation, transcriptional regulation, and cell adhesion/motility. The activities of these proteins are controlled primarily through guanine nucleotide exchange factors (GEFs) that facilitate the exchange of GDP for GTP, promoting the active (GTP-bound) state, and GTPase activating proteins (GAPs) that promote GTP hydrolysis and the inactive (GDP-bound) state (1,2).

The p190 RhoGAP proteins are widely expressed Rho family GAPs. p190-A has been characterized as a tumor suppressor, and research studies have shown that loss or rearrangement of the chromosomal region containing the gene for p190-A is linked to tumor development (3,4). p190-A binds the mitogen-inducible transcription factor TFII-I, sequestering it in the cytoplasm and inhibiting its activity. Phosphorylation of p190-A at Tyr308 reduces its affinity for TFII-I, relieving the inhibition (5). p190-A can also inhibit growth factor-induced gliomas in mice (6) and affect cleavage furrow formation and cytokinesis in cultured cells (7).

Mice lacking p190-B RhoGAP show excessive Rho activation and a reduction in activation of the transcription factor CREB (8). Cells deficient in p190-B display defective adipogenesis (9). There is increasing evidence that p190 undergoes tyrosine phosphorylation, which activates its GAP domain (9-11). Levels of tyrosine phosphorylation are enhanced by Src overexpression (10,11). IGF-I treatment downregulates Rho through phosphorylation and activation of p190-B RhoGAP, thereby enhancing IGF signaling implicated in adipogenesis (9).

  1. Peck, J. et al. (2002) FEBS Lett 528, 27-34.
  2. Moon, S.Y. and Zheng, Y. (2003) Trends Cell Biol 13, 13-22.
  3. Wang, Z. et al. (1996) Cell Growth Differ 7, 123-33.
  4. Tikoo, A. et al. (2000) Gene 257, 23-31.
  5. Jiang, W. et al. (2005) Mol Cell 17, 23-35.
  6. Wolf, R.M. et al. (2003) Genes Dev 17, 476-87.
  7. Su, L. et al. (2003) J Cell Biol 163, 571-82.
  8. Sordella, R. et al. (2002) Dev Cell 2, 553-65.
  9. Sordella, R. et al. (2003) Cell 113, 147-158.
  10. Chang, J.H. et al. (1995) J. Cell Biol. 130, 355-368.
  11. Roof, R.W. et al. (1998) Mol. Cell Biol. 18, 7052-7063.

Pathways & Proteins

Explore pathways + proteins related to this product.

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

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