Western blot analysis of CHIP was performed by loading 40 ug of MCF-7 cell lysates and 3 ul PageRuler Prestained Protein Ladder (Thermo # 26619) per well onto a 10% Tris-HCl polyacrylamide gel. Proteins were transferred to a NC membrane and blocked with 5% skim milk/PBS-T for 1 h at room temperature. CHIP was detected at 35 kDa using a CHIP antibody (Product # PA1-015) at 1:1000 dilution in 5% BSA/PBS-T buffer overnight at 4°C on a rocking platform, followed by a goat anti-rabbit IgG-HRP secondary antibody at a dilution in 5% skim milk/PBS-T of 1:3000 for 2 hour at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). Data courtesy of Dr. Wei Xu at the University of Wisconsin, Madison, WI.
|Tested species reactivity||Human, Mouse|
|Published species reactivity||Rabbit, Rat, Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic peptide corresponding to residues V(218) D E K R K K R D I P D Y L C(232) of human CHIP.|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
PA1-015 detects human and mouse carboxyl terminus of hsc70-interacting protein (CHIP).
PA1-015 has been successfully used in Western blot procedures. By Western blot, this antibody detects an ~35 kDa protein representing CHIP from COS-1 cells overexpressing the human gene and a non-specific band at ~85 kDa.
PA1-015 was used successfully in the western blot analysis of CHIP using human MCF7 cell lysates.
The PA1-015 immunogen is a synthetic peptide corresponding to residues V(218) D E K R K K R D I P D Y L C(232) of human CHIP. This peptide (Cat. # PEP-179) is available for use in neutralization and control experiments.
A recently identified protein, termed carboxyl terminus of hsc70-interacting protein (CHIP), has been shown to interact both with the constitutive form of hsc70 and the stress inducible form, hsp70. This novel 35 kDa cytoplasmic protein has been shown to be highly expressed in striated muscle in vivo. Additional studies have shown that this protein is expressed over a broad range of cultured tissues. Through immunoprecipitation experiments, CHIP has been shown to directly bind to the carboxyl terminus of hsc70 and hsp70 where it decreases ATPase activity and reduces overall chaperone efficiency.
CHIP has also been identified as an important protein in the ubiquitin-proteasome system. CHIP contains a U-box domain and acts as an E3 ubiquitin-ligase in conjunction with hsc70 and hsp90.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Modulation of heme/substrate binding cleft of neuronal nitric-oxide synthase (nNOS) regulates binding of Hsp90 and Hsp70 proteins and nNOS ubiquitination.
PA1-015 was used in western blot to investigate the mechanism for neuronal nitric-oxide synthase activity and degradation
|Peng HM,Morishima Y,Pratt WB,Osawa Y||The Journal of biological chemistry (287:1556)||2012|
Regulation of death-associated protein kinase. Stabilization by HSP90 heterocomplexes.
PA1-015 was used in western blot to study the role of death-associated protein kinase DAPK in apoptosis and cellular homeostasis.
|Zhang L,Nephew KP,Gallagher PJ||The Journal of biological chemistry (282:11795)||2007|
CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-alpha.
PA1-015 was used in western blot to demonstrate the effect of carboxyl terminus of Hsc70-interacting protein on basal and geldanamycin-induced degradation of estrogen receptor-alpha.
|Fan M,Park A,Nephew KP||Molecular endocrinology (Baltimore, Md.) (19:2901)||2005|
CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival.
PA1-015 was used in western blot to investigate the role of CHIP-Hsc70 complex in regulating phosphorylated tau-induced cell death
|Shimura H,Schwartz D,Gygi SP,Kosik KS||The Journal of biological chemistry (279:4869)||2004|
Brain distribution of carboxy terminus of Hsc70-interacting protein (CHIP) and its nuclear translocation in cultured cortical neurons following heat stress or oxygen-glucose deprivation.
PA1-015 was used in immunohistochemistry to investigate the changes in subcellular localization of carboxy terminus of Hsc70-interacting protein (CHIP) in cultured cortical neurons following heat stress or oxygen-glucose deprivation
|Anderson LG,Meeker RB,Poulton WE,Huang DY||Cell stress and chaperones (15:487)||2010|
Inhibition of hsp70 by methylene blue affects signaling protein function and ubiquitination and modulates polyglutamine protein degradation.
PA1-015 was used in blocking/activating experiment to investigate the effect of methylene blue-induced HSP70 inhibition on protein functions and degradation
|Wang AM,Morishima Y,Clapp KM,Peng HM,Pratt WB,Gestwicki JE,Osawa Y,Lieberman AP||The Journal of biological chemistry (285:15714)||2010|
Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.
PA1-015 was used in immunoprecipitation to investigate the interaction between CHIP and Hsc70 as well as Hsp70
|Ballinger CA,Connell P,Wu Y,Hu Z,Thompson LJ,Yin LY,Patterson C||Molecular and cellular biology (19:4535)||1999|