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75706
PREX1 (D8D2K) Rabbit mAb (IHC Specific)

PREX1 (D8D2K) Rabbit mAb (IHC Specific) #75706

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APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 190, 110 Rabbit IgG
IHC-P (paraffin)
Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using PREX1 (D8D2K) Rabbit mAb (IHC Specific).

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IHC-P (paraffin)
Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using PREX1 (D8D2K) Rabbit mAb (IHC Specific).

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IHC-P (paraffin)
Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human lymphoma using PREX1 (D8D2K) Rabbit mAb (IHC Specific).

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IHC-P (paraffin)
Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human melanoma using PREX1 (D8D2K) Rabbit mAb (IHC Specific).

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IHC-P (paraffin)
Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded T-47D (left) and MCF 10A (right) cell pellets using PREX1 (D8D2K) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

A. Solutions and Reagents

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

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

posted February 2010

revised March 2016

Protocol Id: 283

Application Dilutions
Immunohistochemistry (Paraffin) 1:150
Storage:

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

PREX1 (D8D2K) Rabbit mAb (IHC Specific) recognizes endogenous levels of total PREX1 protein by immunohistochemistry. This antibody is not recommended for western blot due to weaker signal in comparison to other antibodies available against PREX1.

Species Reactivity:

Human

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His770 of human PREX1 protein.

Phosphoinositide-3,4,5-triphosphate (PtdIns(3,4,5)P3)-dependent Rac exchanger 1 (PREX1) is a Rac-specific GTP-exchange factor (GEF) regulated by heterotrimeric G-protein β/γ subunits and the lipid second messenger PtdIns(3,4,5)P3 (1-4). PREX1 contains two DEP (Dishevelled, Egl-10, and Pleckstrin homology) domains that coordinate heterotrimeric G-protein signaling. It also contains a Dbl-homology domain, which exhibits Rac-GEF activity, and PH and PDZ domains for interacting with upstream and downstream signaling components (1). Originally shown to modulate cellular migration of neutrophils by Rac2 activation (5-8), it is clear that PREX1 plays a broader role in modulating cell migration. PREX1 promotes metastasis of prostate cancer and melanoma cells, affects endothelial junction integrity, and is required for platelet generation and function (9-14). Research studies suggest that PREX1 plays an essential role in mediating ErbB-dependent signaling events in breast cancer by coordinating Rac activation in response to paracrine signals within the tumor microenvironment. Activation of PREX1 downstream of ErbB3 and EGFR chemokine receptors (CXCR4) promotes Rac activation, increased migration, proliferation, tumorigenesis, and metastasis in breast cancer cells (15,16). Consistent with this observation, deletion of PREX1 expression in mice results in resistance to melanoma metastasis (11). Expression of PREX1 in human tumors transplanted into mice inversely correlates with increased tumor progression and poor survival (15). Additional research studies suggest that PREX Rac-GEF activity is enhanced by phosphorylation in response to growth factors or hormones, and may require coincident dephosphorylation of two PH domain serine residues. The upstream kinases and precise regulatory mechanism remains elusive (15,17).

  1. Welch, H.C. et al. (2002) Cell 108, 809-21.
  2. Hill, K. et al. (2005) J Biol Chem 280, 4166-73.
  3. Mayeenuddin, L.H. and Garrison, J.C. (2006) J Biol Chem 281, 1921-8.
  4. Barber, M.A. et al. (2007) J Biol Chem 282, 29967-76.
  5. Welch, H.C. et al. (2005) Curr Biol 15, 1867-73.
  6. Dong, X. et al. (2005) Curr Biol 15, 1874-9.
  7. Zhao, T. et al. (2007) J Leukoc Biol 81, 1127-36.
  8. Nie, B. et al. (2010) Cell Signal 22, 770-82.
  9. Qin, J. et al. (2009) Oncogene 28, 1853-63.
  10. Wong, C.Y. et al. (2011) J Biol Chem 286, 25813-22.
  11. Lindsay, C.R. et al. (2011) Nat Commun 2, 555.
  12. Qian, F. et al. (2012) Arterioscler Thromb Vasc Biol 32, 768-77.
  13. Naikawadi, R.P. et al. (2012) Circ Res 111, 1517-27.
  14. Campbell, A.D. et al. (2013) PLoS One 8, e53982.
  15. Montero, J.C. et al. (2011) Oncogene 30, 1059-71.
  16. Sosa, M.S. et al. (2010) Mol Cell 40, 877-92.
  17. Montero, J.C. et al. (2013) Cell Signal 25, 2281-9.
Entrez-Gene Id
57580
Swiss-Prot Acc.
Q8TCU6
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.

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75706S		 100 µl (150 sections) $ 263.0

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