This antibody gave a positive signal in Recombinant Hsc70 protein as well as the following whole cell lysates: MCF7; MDA MB 231; HEK293. It also gave a positive result in human kidney FFPE tissue sections.
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.
pH: 7.40 Preservative: 0.02% Sodium azide Constituent: PBS Note: Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
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.
Use a concentration of 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 71 kDa).
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.
Belongs to the heat shock protein 70 family.
The N-terminal 1-386 residues constitute the ATPase domain, while residues 387-646 form the peptide-binding domain.
Phosphorylated upon DNA damage, probably by ATM or ATR. ISGylated.
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.
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
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.
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.