Anti-Hsc70 antibody (ab125307)
Key features and details
- Rabbit polyclonal to Hsc70
- Suitable for: IHC-P, ICC/IF, WB
- Knockout validated
- Reacts with: Human, Recombinant fragment
- Isotype: IgG
Overview
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Product name
Anti-Hsc70 antibody
See all Hsc70 primary antibodies -
Description
Rabbit polyclonal to Hsc70 -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species ICC/IF HumanIHC-P HumanWB HumanRecombinant fragment
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Immunogen
Synthetic peptide conjugated to KLH derived from within residues 550 to the C-terminus of Human Hsc70.
Read Abcam's proprietary immunogen policy -
Positive control
- WB: Wild-type A431 cell lysate. Recombinant Hsc70 protein; A431, HeLa, MCF7, MDA MB 231, and HEK293 whole cell lysates. IHC-P: Human kidney tissue. ICC/IF: HepG2 and HeLa cells.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.4
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration
Concentration information loading...Purity
Immunogen affinity purifiedClonality
PolyclonalIsotype
IgGResearch areas
Associated products
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Compatible Secondaries
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Isotype control
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab125307 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
GuaranteedTested applications are guaranteed to work and covered by our Abpromise guarantee.
PredictedPredicted to work for this combination of applications and species but not guaranteed.
IncompatibleDoes not work for this combination of applications and species.
Application Species ICC/IF HumanIHC-P HumanWB HumanRecombinant fragmentAll applications MouseRatHorseCowChinese hamsterOrangutanApplication Abreviews Notes IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.ICC/IF (1) Use a concentration of 1 µg/ml.WB Use a concentration of 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 71 kDa).Notes IHC-P
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.ICC/IF
Use a concentration of 1 µg/ml.WB
Use a concentration of 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 71 kDa).Target
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Function
Acts as a repressor of transcriptional activation. Inhibits the transcriptional coactivator activity of CITED1 on Smad-mediated transcription. Chaperone. Isoform 2 may function as an endogenous inhibitory regulator of HSC70 by competing the co-chaperones. -
Tissue specificity
Ubiquitous. -
Sequence similarities
Belongs to the heat shock protein 70 family. -
Domain
The N-terminal 1-386 residues constitute the ATPase domain, while residues 387-646 form the peptide-binding domain. -
Post-translational
modificationsPhosphorylated upon DNA damage, probably by ATM or ATR.
ISGylated. -
Cellular localization
Cytoplasm. Melanosome. Localized in cytoplasmic mRNP granules containing untranslated mRNAs. Translocates rapidly from the cytoplasm to the nuclei, and especially to the nucleoli, upon heat shock. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. - Information by UniProt
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Database links
- Entrez Gene: 281831 Cow
- Entrez Gene: 3312 Human
- Entrez Gene: 15481 Mouse
- Entrez Gene: 24468 Rat
- Omim: 600816 Human
- SwissProt: P11142 Human
- SwissProt: P63017 Mouse
- SwissProt: P63018 Rat
see all -
Alternative names
- 2410008N15Rik antibody
- Constitutive heat shock protein 70 antibody
- Epididymis luminal protein 33 antibody
see all
Images
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Western blot - Anti-Hsc70 antibody (ab125307)All lanes : Anti-Hsc70 antibody (ab125307) at 1 µg/ml
Lane 1 : Wild-type A431 cell lysate
Lane 2 : HSPA8 knockout A431 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MCF7 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 71 kDa
Observed band size: 73 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab125307 observed at 73 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab125307 was shown to react with HSPA8 in wild-type A431 cells in western blot. Loss of signal was observed when HSPA8 knockout sample was used. Wild-type and HSPA8 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab125307 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.
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Immunocytochemistry/ Immunofluorescence - Anti-Hsc70 antibody (ab125307)ab125307 staining HSC70 in HeLa cells treated with (S)-(+)-ibuprofen (ab141015), by ICC/IF. Increase of HSC70 nuclear expression correlates with increased concentration of (S)-(+)-ibuprofen, as described in literature.
The cells were incubated at 37°C for 1 hour in media containing different concentrations of ab141015 ((S)-(+)-ibuprofen) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab125307 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue. -
Western blot - Anti-Hsc70 antibody (ab125307)All lanes : Anti-Hsc70 antibody (ab125307) at 1 µg/ml
Lane 1 : Recombinant Protein:Hsc70 (Active)
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 3 : MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 71 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?
Additional bands at: 46 kDa, 52 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 4 minutes -
Immunocytochemistry/ Immunofluorescence - Anti-Hsc70 antibody (ab125307)ICC/IF image of ab125307 stained HepG2 cells. The cells were 4% formaldehyde 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 (ab125307, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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% formaldehyde fixed (10 min) HeLa cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa, HepG2 and MCF7 cells at 1µg/ml. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsc70 antibody (ab125307)IHC image of ab125307 staining in human kidney 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 ab1253007, 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.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Protocols
Datasheets and documents
References (0)
ab125307 has not yet been referenced specifically in any publications.
Images
-
Western blot - Anti-Hsc70 antibody (ab125307)All lanes : Anti-Hsc70 antibody (ab125307) at 1 µg/ml
Lane 1 : Wild-type A431 cell lysate
Lane 2 : HSPA8 knockout A431 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MCF7 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 71 kDa
Observed band size: 73 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab125307 observed at 73 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab125307 was shown to react with HSPA8 in wild-type A431 cells in western blot. Loss of signal was observed when HSPA8 knockout sample was used. Wild-type and HSPA8 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab125307 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.
-
Immunocytochemistry/ Immunofluorescence - Anti-Hsc70 antibody (ab125307)
ab125307 staining HSC70 in HeLa cells treated with (S)-(+)-ibuprofen (ab141015), by ICC/IF. Increase of HSC70 nuclear expression correlates with increased concentration of (S)-(+)-ibuprofen, as described in literature.
The cells were incubated at 37°C for 1 hour in media containing different concentrations of ab141015 ((S)-(+)-ibuprofen) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab125307 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue. -
Western blot - Anti-Hsc70 antibody (ab125307)All lanes : Anti-Hsc70 antibody (ab125307) at 1 µg/ml
Lane 1 : Recombinant Protein:Hsc70 (Active)
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 3 : MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 71 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?
Additional bands at: 46 kDa, 52 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 4 minutes
-
Immunocytochemistry/ Immunofluorescence - Anti-Hsc70 antibody (ab125307)ICC/IF image of ab125307 stained HepG2 cells. The cells were 4% formaldehyde 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 (ab125307, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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% formaldehyde fixed (10 min) HeLa cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa, HepG2 and MCF7 cells at 1µg/ml.
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hsc70 antibody (ab125307)IHC image of ab125307 staining in human kidney 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 ab1253007, 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.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
