Immunofluorescence analysis of Phospho-HSP27 pSer15 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 pSer15 Rabbit Polyclonal Antibody (PA1018) 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||Rabbit, Rat, Human, Not Applicable|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic phosphopeptide corresponding to the residues L(10) L R G P (pS) W D P F R C(21) of human HSP27.|
|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 *|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||1:50 - 1:200|
|Immunofluorescence (IF)||1:50 - 1:200|
|Immunohistochemistry (IHC)||1:50 - 1:200|
|Immunoprecipitation (IP)||3 µg|
|Western Blot (WB)||1:100 - 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.
PA1-018 detects phosphorylated heat shock protein 27(hsp27) from rat and human tissue tissues.
PA1-018 has been successfully used in Western blot, immunoprecipitation, immunofluorscence, immunocytochemistry and immunohistochemistry procedures. Immunocytochemical staining of phospho-hsp27 (Ser15) in HeLa cells with this antibody results in cytoplasmic staining.
The PA1-018 immunogen is a synthetic phosphopeptide corresponding to the residues L(10) L R G P (pS) W D P F R C(21) of human HSP27. The immunizing peptide is 81% homologous in mice, rats, and hamsters. This peptide (Cat. # PEP-187) is available for use in neutralization and control experiments.
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.
Cell stress promotes the association of phosphorylated HspB1 with F-actin.
PA1-018 was used in immunocytochemistry to assess the association of phosphorylated HspB1 with F-actin promoted by cell stress
|Clarke JP,Mearow KM||PloS one (8:null)||2013|
Distension of the uterus induces HspB1 expression in rat uterine smooth muscle.
PA1-018 was used in immunocytochemistry to study the expression of HspB1 in rat uterine smooth muscle
|White BG,MacPhee DJ||American journal of physiology. Regulatory, integrative and comparative physiology (301:R1418)||2011|
Hsp27 and axonal growth in adult sensory neurons in vitro.
PA1-018 was used in immunocytochemistry to demonstrate the interaction between actin and Hsp27 and its role in neurite outgrowth.
|Williams KL,Rahimtula M,Mearow KM||BMC neuroscience (6:null)||2005|
The hsp27kD heat shock protein and p38-MAPK signaling are required for regular epidermal differentiation.
PA1-018 was used in western blot to investigate the important role of hsp27 protein in epidermal differentiation
|Jonak C,Mildner M,Klosner G,Paulitschke V,Kunstfeld R,Pehamberger H,Tschachler E,Trautinger F||Journal of dermatological science (61:32)||2011|