The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 1 µg/ml. Detects a band of approximately 27 kDa (predicted molecular weight: 23 kDa).
Involved in stress resistance and actin organization.
Detected in all tissues tested: skeletal muscle, heart, aorta, large intestine, small intestine, stomach, esophagus, bladder, adrenal gland, thyroid, pancreas, testis, adipose tissue, kidney, liver, spleen, cerebral cortex, blood serum and cerebrospinal fluid. Highest levels are found in the heart and in tissues composed of striated and smooth muscle.
Involvement in disease
Defects in HSPB1 are the cause of Charcot-Marie-Tooth disease type 2F (CMT2F) [MIM:606595]. CMT2F is a form of Charcot-Marie-Tooth disease, the most common inherited disorder of the peripheral nervous system. Charcot-Marie-Tooth disease is classified in two main groups on the basis of electrophysiologic properties and histopathology: primary peripheral demyelinating neuropathy or CMT1, and primary peripheral axonal neuropathy or CMT2. Neuropathies of the CMT2 group are characterized by signs of axonal regeneration in the absence of obvious myelin alterations, normal or slightly reduced nerve conduction velocities, and progressive distal muscle weakness and atrophy. Nerve conduction velocities are normal or slightly reduced. CMT2F onset is between 15 and 25 years with muscle weakness and atrophy usually beginning in feet and legs (peroneal distribution). Upper limb involvement occurs later. CMT2F inheritance is autosomal dominant. Defects in HSPB1 are a cause of distal hereditary motor neuronopathy type 2B (HMN2B) [MIM:608634]. Distal hereditary motor neuronopathies constitute a heterogeneous group of neuromuscular disorders caused by selective impairment of motor neurons in the anterior horn of the spinal cord, without sensory deficit in the posterior horn. The overall clinical picture consists of a classical distal muscular atrophy syndrome in the legs without clinical sensory loss. The disease starts with weakness and wasting of distal muscles of the anterior tibial and peroneal compartments of the legs. Later on, weakness and atrophy may expand to the proximal muscles of the lower limbs and/or to the distal upper limbs.
Belongs to the small heat shock protein (HSP20) family.
Phosphorylated in MCF-7 cells on exposure to protein kinase C activators and heat shock.
Cytoplasm. Nucleus. Cytoplasm > cytoskeleton > spindle. Cytoplasmic in interphase cells. Colocalizes with mitotic spindles in mitotic cells. Translocates to the nucleus during heat shock and resides in sub-nuclear structures known as SC35 speckles or nuclear splicing speckles.
heat shock protein family B (small) member 1 antibody
Hsp 25 antibody
HSP 27 antibody
Hsp 28 antibody
Hsp B1 antibody
Stress responsive protein 27 antibody
Stress-responsive protein 27 antibody
Western blot - Anti-Hsp27 antibody (ab78806)
Predicted band size : 23 kDa
Lane 1: Wild type HAP1 whole cell lysate (20 µg) Lane 2: Hsp27 knockout HAP1 whole cell lysate (20 µg) Lane 3: HeLa whole cell lysate (20 µg) Lane 4: MCF7 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab78806 observed at 27 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab78806 was shown to specifically react with Hsp27 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when Hsp27 knockout samples were used. Wild-type and HSPB1 knockout samples were subjected to SDS-PAGE. Ab78806 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.
ICC/IF image of ab78806 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normaL goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab78806, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2 and MCF7 cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa and MCF7 cells at 1µg/ml.
Western blot - Hsp27 antibody (ab78806)
All lanes : Anti-Hsp27 antibody (ab78806) at 1 µg/ml
Lane 1 : Human skeletal muscle tissue lysate - total protein (ab29330) Lane 2 : Human heart tissue lysate - total protein (ab29431) Lane 3 : Human liver tissue lysate - total protein (ab29889)
Lysates/proteins at 10 µg per lane.
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 23 kDa Observed band size : 27 kDa (why is the actual band size different from the predicted?) Additional bands at : 30 kDa,60 kDa. We are unsure as to the identity of these extra bands.Hsp27 contains a number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.
IHC image of Hsp27 staining in human cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab78806, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.