Immunofluorescence analysis of Phospho-HSP27 pSer85 was done on 70% confluent log phase HeLa cells. The cells were treated with 25ug of Anisomycin for 30 minutes and cell 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 with of Phospho-HSP27 pSer85 Rabbit Polyclonal Antibody (PA1005) at 2ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is untreated cell with no signal. Panel f is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Rat|
|Published species reactivity||Rat, Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic phosphopeptide corresponding to residues R(82) Q L S(p) S G V S E I R(92) C of rat HSP27.|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 30mg/ml BSA|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||15 -25 µg/ml|
|Immunofluorescence (IF)||15 - 25 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:100|
|Immunoprecipitation (IP)||3 µg|
|Western Blot (WB)||0.25 - 2 µg/ml|
* 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 2 publications below|
PA1-005 detects phosphorylated heat shock protein 27 (HSP27) from human and rat tissue samples. This antibody does not detect the unphosphorylated form of the protein.
PA1-005 has been successfully used in Western blot, immunoprecipitation, and immunofluorescence procedures. By Western blot, this antibody detects an ~27 kDa protein representing phosphorylated HSP27(Ser85) from rat skeletal muscle extracts.
The PA1-005 immunogen is a synthetic phosphopeptide corresponding to residues R(82) Q L S(p) S G V S E I R(92) C of rat HSP27. This sequence is completely conserved in mouse.
HSP27 also referred to as the Estrogen-Regulated 24K protein and HSP28, is one of several small heat shock proteins (HSP) produced by all organisms studied. HSP27 synthesis is induced by elevated temperature, as well as estrogen in hormone responsive cells. Interestingly, human HSP27 also shares greater than 50% homology with low molecular weight Drosophila HSP's and mammalian a-crystalline lens protein. Because of the estrogen responsive nature of HSP27, this protein has been studied extensively in human estrogen responsive tissues such as cervix, endometrium and breast tissue. This work has led to the suggestion that HSP27 may be a useful marker in classifying various hormone sensitive tumors.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Increased cell surface Fas expression is necessary and sufficient to sensitize lung fibroblasts to Fas ligation-induced apoptosis: implications for fibroblast accumulation in idiopathic pulmonary fibrosis.
PA1-005 was used in western blot to investigate the role of fas cell surface expression in fas ligation-induced apoptosis in fibroblasts
|Wynes MW,Edelman BL,Kostyk AG,Edwards MG,Coldren C,Groshong SD,Cosgrove GP,Redente EF,Bamberg A,Brown KK,Reisdorph N,Keith RC,Frankel SK,Riches DW||Journal of immunology (Baltimore, Md. : 1950) (187:527)||2011|
Neuroprotective preconditioning of rat brain cultures with ethanol: potential transduction by PKC isoforms and focal adhesion kinase upstream of increases in effector heat shock proteins.
PA1-005 was used in western blot to investigate the effect of preconditioning hippocampal-entorhinocortical on upregulation/activation of PKC isoforms and FAK
|Sivaswamy S,Neafsey EJ,Collins MA||The European journal of neuroscience (32:1800)||2010|