Western blot analysis of extracts from RD and T-47D cells using CRP2 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). The differential in CRP2 expression between RD and T-47D cells is consistent with their reported molecular expression profiles (www.depmap.org), confirming specificity of the antibody for CRP2.
Immunoprecipitation of CRP2 protein from RD cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is CRP2 Antibody. Western blot analysis was performed using CRP2 Antibody. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
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.
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.
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.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
CRP2 Antibody recognizes endogenous levels of total CRP2 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly89 of human CRP2 protein. Antibodies are purified by peptide affinity chromatography.
CRP2 is a LIM domain containing protein that is expressed from the CSRP2 gene. It was first described to be a differentially regulated and preferentially expressed protein in aortic smooth muscle cells (1). It plays a role in development of the vasculature in embryogenesis (2). It was also established that CRP2 is expressed exclusively in stellate cells in the liver, being absent from hepatocytes, sinusoidal endothelial cells, and Kupffer cells. Upregulation of CRP2 was observed to occur upon early activation in the myofibroblastic program of stellate cells, and is now thought to be involved in the development of liver fibrosis (3).
More recently, CRP2 has been shown to be an invadopodia actin bundling protein. Invadopodia are actin-rich membrane protrusions that direct extracellular matrix degradation that are believed to facilitate tumor cell invasion. CRP2 has been shown to be upregulated in breast tumors (4), and in B-cell acute lymphoblastic leukemia high CRP2 expression has been associated with poor outcome (5). The proximal promoter of the CSRP2 gene has been shown to possess two hypoxia responsive elements that are targeted by HIF-1α. A model now is proposed whereby the CSRP2 gene is a direct target of HIF-1 which facilitates hypoxia-induced breast cancer cell invasion through increased invadopodia formation (6).
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