Immunofluorescence analysis of HSC70 Interacting Protein (HIP) was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled HSC70 Interacting Protein HIP (2G6) Mouse Monoclonal Antibody (MA3413) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Chicken, Human, Mouse, Rabbit|
|Published species reactivity||Mouse|
|Host / Isotype||Mouse / IgG|
|Immunogen||Recombinant human Hip protein.|
|Storage buffer||ascites diluted in PBS|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||1:20|
|Immunoprecipitation (IP)||Assay dependent|
|Western Blot (WB)||1:250-1:1,000|
* 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.
|Western Blot (WB)||See 1 publications below|
MA3-413 detects heat shock cognate protein 70 kDa (HSC70)-interacting protein (Hip) from human, mouse, chicken and rabbit samples.
MA3-413 has been successfully used in Western blot, immunofluorescence, and immunoprecipitation procedures. By Western blot, this antibody detects a 48 kDa protein representing Hip from rabbit reticulocyte lysate.
The MA3-413 antigen is recombinant human Hip protein.
Steroid receptors are ligand-dependent intracellular proteins that stimulate transcription of specific genes by binding to specific DNA sequences following activation by the appropriate hormone. Prior to activation, steroid receptors associate with a number of different proteins in both a stable and transient fashion. Steroid receptor complex proteins include heat shock proteins (HSP90, HSP70), immunosuppressant binding proteins called immunophilins (the FK506 binding proteins, FKBP52 and FKBP54 and the cyclosporin binding protein, CyP-40) and three other proteins termed p23, p60 and heat shock cognate protein 70 kDa (HSC70)-interacting protein (Hip), also known as p48. Hip, along with HSP70 and p60, combine with steroid receptors as transient intermediate complex members.
In vitro studies have suggested that Hip transiently interacts with progesterone receptor in a cell free system prior to hormone binding competency and dissociates from the receptor complex with the binding of an HSP90:Immunophilin:p23 subcomplex which then results in a hormone-binding-competent receptor complex. While neither its exact function nor mechanism of action have been identified, Hip appears to be an important factor in steroid receptor function. Hip also appears to regulate the function of HSC70 in an ATP hydrolysis and HSP40 dependent fashion.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Differential effects of the hsp70-binding protein BAG-1 on glucocorticoid receptor folding by the hsp90-based chaperone machinery.
MA3-413 was used in western blot to investigate the different roles of the hsp70-binding protein BAG-1 during glucocorticoid receptor folding
|Kanelakis KC,Morishima Y,Dittmar KD,Galigniana MD,Takayama S,Reed JC,Pratt WB||The Journal of biological chemistry (274:34134)||1999|