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Signal Transduction Protein Trafficking Chaperones Heat Shock Proteins
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)

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  • Certificate of Compliance
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Western blot - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
  • Flow Cytometry - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
  • Western blot - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
  • Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR5477] to Hsp27 - BSA and Azide free
  • Suitable for: WB, Flow Cyt, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

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PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
Protein
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Recombinant Human Hsp27 (phospho S82) protein (ab113185)

View more associated products

Overview

  • Product name

    Anti-Hsp27 antibody [EPR5477] - BSA and Azide free
    See all Hsp27 primary antibodies
  • Description

    Rabbit monoclonal [EPR5477] to Hsp27 - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, Flow Cyt, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    A synthetic peptide corresponding to residues in Human Hsp27

  • Positive control

    • WB: HeLa, HAP1, COS-1, BxPC-3, and HT-1376 cell lysates. ICC/IF: HeLa cells. Flow Cyt: HAP1 cells.
  • General notes

    ab229442 is the carrier-free version of ab109376. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    ab229442 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C. Do Not Freeze.
  • Storage buffer

    pH: 7.20
    Constituent: PBS
  • Carrier free

    Yes
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR5477
  • Isotype

    IgG
  • Research areas

    • Signal Transduction
    • Protein Trafficking
    • Chaperones
    • Heat Shock Proteins
    • Cancer
    • Tumor biomarkers
    • Other
    • Cardiovascular
    • Atherosclerosis
    • Ischemia / Reperfusion
    • Cardiovascular
    • Heart
    • Contractility
    • Contractile Proteins
    • Actins

Associated products

  • Alternative Versions

    • Anti-Hsp27 antibody [EPR5477] (ab109376)
    • Alexa Fluor® 647 Anti-Hsp27 antibody [EPR5477] (ab194078)
    • HRP Anti-Hsp27 antibody [EPR5477] (ab194079)
    • Alexa Fluor® 594 Anti-Hsp27 antibody [EPR5477] (ab215328)
  • Compatible Secondaries

    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human HSPB1 (Hsp27) knockout HeLa cell line (ab261738)
  • KO cell lysates

    • Human HSPB1 (Hsp27) knockout HeLa cell lysate (ab256945)
  • Positive Controls

    • HeLa nuclear extract lysate (ab14655)
  • Recombinant Protein

    • Recombinant Human Hsp27 (phospho S82) protein (ab113185)

Applications

Our Abpromise guarantee covers the use of ab229442 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use at an assay dependent concentration. Predicted molecular weight: 23 kDa.
Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG (Low endotoxin, Azide free), is suitable for use as an isotype control with this antibody.

ICC/IF Use at an assay dependent concentration.

Target

  • Function

    Involved in stress resistance and actin organization.
  • Tissue specificity

    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.
  • Sequence similarities

    Belongs to the small heat shock protein (HSP20) family.
  • Post-translational
    modifications

    Phosphorylated in MCF-7 cells on exposure to protein kinase C activators and heat shock.
  • Cellular localization

    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.
  • Target information above from: UniProt accession P04792 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 3315 Human
    • Entrez Gene: 15507 Mouse
    • Entrez Gene: 24471 Rat
    • Omim: 602195 Human
    • SwissProt: P04792 Human
    • SwissProt: P14602 Mouse
    • SwissProt: P42930 Rat
    • Unigene: 520973 Human
    • Unigene: 13849 Mouse
    • Unigene: 3841 Rat
    see all
  • Alternative names

    • Heat shock 27kDa protein antibody
    • 28 kDa heat shock protein antibody
    • CMT2F antibody
    • DKFZp586P1322 antibody
    • epididymis secretory protein Li 102 antibody
    • Estrogen regulated 24 kDa protein antibody
    • Estrogen-regulated 24 kDa protein antibody
    • Heat shock 25kDa protein 1 antibody
    • Heat shock 27 kDa protein antibody
    • Heat shock 27kD protein 1 antibody
    • Heat shock 27kDa protein 1 antibody
    • Heat shock 28kDa protein 1 antibody
    • Heat Shock Protein 27 antibody
    • Heat shock protein beta 1 antibody
    • Heat shock protein beta-1 antibody
    • heat shock protein family B (small) member 1 antibody
    • HEL-S-102 antibody
    • HMN2B antibody
    • HS.76067 antibody
    • Hsp 25 antibody
    • HSP 27 antibody
    • Hsp 28 antibody
    • Hsp B1 antibody
    • Hsp25 antibody
    • HSP27 antibody
    • Hsp28 antibody
    • HspB1 antibody
    • HSPB1_HUMAN antibody
    • SRP27 antibody
    • Stress responsive protein 27 antibody
    • Stress-responsive protein 27 antibody
    see all

Images

  • Western blot - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
    Western blot - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
    All lanes : Anti-Hsp27 antibody [EPR5477] (ab109376) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : HSPB1 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 23 kDa
    Observed band size: 23 kDa



    This data was developed using the same antibody clone in a different buffer formulation (ab109376).

      Lanes 1- 2: Merged signal (red and green). Green - ab109376 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab109376 was shown to react with Hsp27 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261738 (knockout cell lysate ab256945) was used. Wild-type HeLa and HSPB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109376 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Flow Cytometry - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
    Flow Cytometry - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)

    Overlay histogram showing HAP1 wildtype (green line) and HAP1-HSPB1 knockout cells (red line) stained with ab109376. The cells were fixed with 4% formaldehyde (10 min)  and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab109376, 1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) presorbed (ab150081) at 1/2000 dilution for 30 min at 22°C.

    A rabbit IgG isotype control antibody  (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-HSPB1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

    Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109376).

  • Immunocytochemistry/ Immunofluorescence - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
    Immunocytochemistry/ Immunofluorescence - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)

    Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling Hsp27 with purified ab109376 at 1/500. Cells were fixed with 100% methanol. ab150077, Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

    Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109376).

  • Western blot - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
    Western blot - Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
    All lanes : Anti-Hsp27 antibody [EPR5477] (ab109376) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : Hsp27 knockout HAP1 whole cell lysate
    Lane 3 : HeLa whole cell lysate
    Lane 4 : MCF7 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 23 kDa



    This WB data was generated using the same anti-Hsp27 antibody clone, EPR5477, in a different buffer formulation (cat# ab109376).

    Lanes 1 - 4: Merged signal (red and green). Green - ab109376 observed at 27 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab109376 was shown to specifically react with Hsp27 in wild-type cells as signal was lost in Hsp27 knockout HEP1 cells. Wild-type and Hsp27 knockout samples were subjected to SDS-PAGE.  Ab109376 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution 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.

  • Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)
    Anti-Hsp27 antibody [EPR5477] - BSA and Azide free (ab229442)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

    • Datasheet

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

  • References (0)

    Publishing research using ab229442? Please let us know so that we can cite the reference in this datasheet.

    ab229442 has not yet been referenced specifically in any publications.

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