Cat. # | Size | Qty. | Price |
---|---|---|---|
75468S | 100 µl |
|
REACTIVITY | H M |
SENSITIVITY | Endogenous |
MW (kDa) | 20 |
SOURCE | Rabbit |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
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: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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: When using primary antibodies produced in rabbit to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) as a secondary antibody to minimize interference produced by denatured rabbit heavy chain. For proteins with a molecular weight in the range of 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured mouse light chain.
When using primary antibodies produced in mouse to detect proteins with a molecular weight in the range of 50 kDa, we recommend using Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) (#58802) as a secondary antibody to minimize interference produced by denatured mouse heavy chain.
posted December 2007
revised October 2021
Protocol Id: 27
Human, Mouse
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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