Detects a ~70kDa protein corresponding to the molecular mass of inducible Hsp70 on SDS PAGE immunoblots. May cross-react with Hsc70 at lower dilutions.
Recommended Dilutions
1:25,000 (ECL) (WB), 1:100 (IP)
Form
Rabbit antiserum
Storage Buffer
Rabbit antiserum
Concentration
N/A
Certificate of Analysis
A 1:10,000 dilution of SPC-103 was sufficient for detection of hsp70 in 20μg of HeLa cell lysate by ECL immunoblot analysis.
Storage Temp
-20°C
Shipping Temp
Blue Ice or 4°C
This antibody is available conjugated to several dyes. Please select one of the conjugates from the lists below:
ATTO Conjugates:
ATTO-Conjugates.pdf
Enzyme & Fluorescent Conjugates:
Enzyme-Fluorescent-Conjugate.pdf
ATTO Conjugates:
Atto 390:
ATTO 390 is a novel fluorescent label with a coumarin structure. Characteristic features of the label are high fluorescence quantum yield, large Stokes-shift, good photostability, and low molecular weight. ATTO 390 is moderately hydrophilic. Its fluorescence can be excited efficiently in the range 360 - 410 nm. A useful excitation source is, e.g., a Mercury Arc Lamp with its lines at 365 nm and 405 nm.
ATTO Conjugates:
Atto 488:
ATTO 488 is a fluorescent label with excellent water solubility. Characteristic features of the label are strong absorption, high fluorescence quantum yield, high photostability, and very little triplet formation. Thus ATTO 488 is highly suitable for single-molecule detection applications and high-resolution microscopy such as PALM, dSTORM, STED etc. Additionally the dye highly qualifies to be applied in flow cytometry (FACS), fluorescence in-situ hybridization (FISH) and many more. The fluorescence is excited most efficiently in the range 480 - 515 nm. For instance the 488 nm line of the Argon-Ion laser is very suitable for excitation
ATTO Conjugates:
Atto 565:
ATTO 565 is a novel fluorescent label belonging to the class of Rhodamine dyes. Characteristic features of the label are strong absorption, high fluorescence quantum yield, and high thermal and photo-stability. Thus ATTO 565 is highly suitable for single-molecule detection applications and high-resolution microscopy such as PALM, dSTORM, STED etc. Additionally the dye highly qualifies to be applied in flow cytometry (FACS), fluorescence in-situ hybridization (FISH) and many more. The dye is moderately hydrophilic.
ATTO Conjugates:
Atto 594:
ATTO 594 is a novel fluorescent label belonging to the class of Rhodamine dyes. Characteristic features of the label are strong absorption, high fluorescence quantum yield, high thermal and photo-stability, and excellent water solubility. Thus ATTO 590 is highly suitable for single-molecule detection applications and high-resolution microscopy such as PALM, dSTORM, STED etc. Additionally the dye highly qualifies to be applied in flow cytometry (FACS), fluorescence in-situ hybridization (FISH) and many more. Due to particular substituents ATTO 594 is very hydrophilic. After coupling to a substrate the label carries a net electrical charge of -1.
ATTO Conjugates:
Atto 633:
ATTO 633 belongs to a new generation of fluorescent labels for the red spectral region. Characteristic features of the label are strong absorption, high fluorescence quantum yield, and high thermal and photo-stability. Thus ATTO 633 is highly suitable for single-molecule detection applications and high-resolution microscopy such as PALM, dSTORM, STED etc. Additionally the dye highly qualifies to be applied in flow cytometry (FACS), fluorescence in-situ hybridization (FISH) and many more. In common with most ATTO-labels, absorption and fluorescence are independent of pH, at least in the range of pH 2 to 11, used in typical applications. ATTO 633 is a cationic dye. After coupling to a substrate the label carries a net electrical charge of +1.
ATTO Conjugates:
Atto 655:
ATTO 655 belongs together with ATTO 680 und ATTO 700 to a new generation of fluorescent labels. Characteristic features of the label are strong absorption, high fluorescence quantum yield, high thermal and photo-stability, exceptionally high stability towards atmospheric ozone, and very good water solubility. Thus ATTO 655 is highly suitable for single-molecule detection applications and high-resolution microscopy such as PALM, dSTORM, STED etc. Additionally the dye highly qualifies to be applied in flow cytometry (FACS), fluorescence in-situ hybridization (FISH) and many more. ATTO 655 is a zwitterionic dye. After coupling to a substrate the dye moiety is electrically neutral. ATTO 655 is a strong electron acceptor. Its fluorescence is efficiently quenched by electron donors like guanine, tryptophan, etc. The fluorescence is excited most efficiently in the range 640 - 660 nm. A suitable excitation source is the 647 nm line of the Krypton-Ion laser or a diode-laser emitting at 650 nm.
ATTO Conjugates:
Atto 680:
ATTO 680 belongs together with ATTO 655 and ATTO 700 to a new generation of fluorescent labels. Characteristic features of the label are strong absorption, high fluorescence quantum yield, good water solubility, and high thermal and photo-stability. ATTO 680 is a zwitterionic dye. After coupling to a substrate the dye moiety is electrically neutral. ATTO 680 is a strong electron acceptor. Its fluorescence is efficiently quenched by electron donors like guanine, tryptophan, etc. The fluorescence is excited most efficiently in the range 645 - 695 nm. A suitable excitation source for the dye is the Krypton-Ion laser using the 676 nm line or a diode-laser emitting at 670 nm.
ATTO Conjugates:
Atto 700:
ATTO 700 belongs together with ATTO 655 and ATTO 680 to a new generation of fluorescent labels. Characteristic features of the label are strong absorption, high fluorescence quantum yield, good water solubility, and high thermal and photo-stability. ATTO 700 is a zwitterionic dye. After coupling to a substrate the dye moiety is electrically neutral. ATTO 700 is a strong electron acceptor. Its fluorescence is efficiently quenched by electron donors like guanine, tryptophan, etc. The fluorescence is excited most efficiently in the range 670 - 715 nm.
ATTO Conjugates:
PE/Atto 594:
ATTO 594 is a novel fluorescent label belonging to the class of Rhodamine dyes. Characteristic features of the label are strong absorption, high fluorescence quantum yield, high thermal and photo-stability, and excellent water solubility. Thus ATTO 590 is highly suitable for single-molecule detection applications and high-resolution microscopy such as PALM, dSTORM, STED etc. Additionally the dye highly qualifies to be applied in flow cytometry (FACS), fluorescence in-situ hybridization (FISH) and many more. Due to particular substituents ATTO 594 is very hydrophilic. After coupling to a substrate the label carries a net electrical charge of -1.
Enzyme & Fluorescent Conjugates:
Alkaline Phosphatase:
Alkaline phosphatase (AP) is a hydrolyase enzyme that is frequently conjugated to antibodies for use in immunoassays. PNPP is a popular colorimetric substrate for AP; the product of the reaction, p-nitrophenol, can be read at 405nM. Fluorogenic substrates (e.g. 4-methylumbelliferyl phosphate; MUP) may also be employed.
Enzyme & Fluorescent Conjugates:
APC:
Allophycocyanin (APC) is a large 105kDa fluorescent phycobiliprotein commonly used in FACS analysis. It absorbs at 650nm and emits at 662nm.
Enzyme & Fluorescent Conjugates:
Biotin:
The attachment of biotin to biomolecules is an important laboratory technique. Biotin binds to the tetrameric avidin proteins, including streptavidin and neutravidin, with exceptionally high affinity, and this interaction is exploited in various applications such as western blotting, immunohisthochemistry and ELISA. The biotin in the kit has an extended linker to facilitate molecular interactions.
Enzyme & Fluorescent Conjugates:
Fluorescein:
Fluorescein (FITC) is a fluorophore widely used in biological analysis, with a pH dependent absorption and emission spectrum. In water it shows an absorption maximum at 494 nm and an emission maximum of 520 nm.
Enzyme & Fluorescent Conjugates:
HRP:
The enzyme horseradish peroxidase (HRP) is known for its ability to amplify a weak signal and increase dectectability of a target molecule. It is an alpha-helical protein which binds heme as a cofactor. It is a 44,173.9 dalton glycoprotein with 6 lysine residues which can be conjugated to a labelled molecule. It produces a coloured, fluorimetric or luminescent derivative of the labeled molecule.
Enzyme & Fluorescent Conjugates:
PerCP:
PerCP (Peridinin-Chlorophyll-Protein Complex) is a small 35kDa phycobiliprotein isolated from red algae. It has a strong absorption peak at 482nm and a secondary absorption peak at 442nm. PerCP has a large stokes shift (195nm) with its maximum emission peak at 677nm. PerCP is commonly used for fluorescent immunolabeling, particularly in applications involving fluorescent-activated cell sorting.
Enzyme & Fluorescent Conjugates:
RPE:
R-Phycoerythrin, or RPE, is useful in the laboratory as a fluorescence-based indicator for the presence of cyanobacteria and for labeling antibodies in a technique called immunofluorescence, among other applications. It is among the brightest fluorescent dyes ever identified. It is predominantly produced by red algae and is made up of at least three different subunits and varies according to the species of algae that produces it.
Enzyme & Fluorescent Conjugates:
Streptavidin:
Streptavidin is a 53kDa protein purified from Streptomyces avidinii, which has widespread applications due to its very high affinity to the vitamin biotin. Streptavidin is a tetrameric molecule, composed of 4 13kDa monomers, each of which can bind a molecule of biotin.
Western blot analysis of Hsp70 in Pam212 cells using a 1:1000 dilution of SPC-103.
Western blot analysis of Hsp70 in multiple human and rat brain cell lysates using a 1:1000 dilution of SPC-103.
Hap70 visualized using SPC-103, tested on Bouin's fixed paraffin-embedded backskin sections of transgenic mice.
Courtesy of Dr. Turksen, Ottawa Hospital Research Institute, Canada.
Research Background
Hsp70 genes encode abundant heat-inducible 70-kDa hsps (hsp70s). In most eukaryotes hsp70 genes exist as part of a multigene family. They are found in most cellular compartments of eukaryotes including nuclei, mitochondria, chloroplasts, the endoplasmic reticulum and the cytosol, as well as in bacteria. The genes show a high degree of conservation, having at least 5O% identity (1, 2). The N-terminal two thirds of hsp70s are more conserved than the C-terminal third. Hsp70 binds ATP with high affinity and possesses a weak ATPase activity which can be stimulated by binding to unfolded proteins and synthetic peptides (3). When hsc70 (constitutively expressed) present in mammalian cells was truncated, ATP binding activity was found to reside in an N-terminal fragment of 44 kDa which lacked peptide binding capacity. Polypeptide binding ability therefore resided within the C-terminal half (4). The structure of this ATPbinding domain displays multiple features of nucleotide binding proteins (5).
All hsp70s, regardless of location, bind proteins, particularly unfolded ones. The molecular chaperones of the hsp70 family recognize and bind to nascent polypeptide chains as well as partially folded intermediates of proteins preventing their aggregation and misfolding. The binding of ATP triggers a critical conformational change leading to the release of the bound substrate protein (6). The universal ability of hsp70s to undergo cycles of binding to and release from hydrophobic stretches of partially unfolded proteins determines their role in a great variety of vital intracellular functions such as protein synthesis, protein folding and oligomerization and protein transport.
References
1. Welch W.J. and Suhan J.P. (1986) J.Cell Biol. 103: 2035-2050.
2. Boorstein W. R., Ziegelhoffer T. & Craig E. A. (1993) J. Mol. Evol. 38(1): 1-17.
3. Rothman J. (1989) Cell 59: 591 -601.
4. DeLuca-Flaherty et al. (1990) Cell 62: 875-887.
5. Bork P., Sander C. & Valencia A. (1992) Proc. Nut1 Acad. Sci. USA 89: 7290-7294.
6. Fink A.L. (1999) Physiol. Rev. 79: 425-449.
7. Hung T.H., et al. (2001) Am J Pathol. 159: 1031-1043.
8. Locke M. (2000) Cell Stress & Chaperones 5: 45-51.
9. Ianaro A., et al. (2001) FEBS Lett. 508: 61-66.
10.Trentin G.A. et al. (2001) J Biol Chem. 276: 13087- 13095.
Cited References
1. Anne Bailey and Lorne J. Hofseth. A method to enhance the sensitivity and reproducibility of immunohistochemistry http://www.prohisto.com/pdfs/Hofseth_Manuscript-81.pdf
2. Jennifer S. Ings, Ken D. Oakes, Mathilakath M. Vijayan, Mark R. Servos. Temporal changes in stress and tissue-specific metabolic responses to municipal wastewater effluent exposure in rainbow trout. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. Available online 1 May 2012. http://dx.doi.org/10.1016/j.cbpc.2012.04.002
3. Donglei Zhang, Fabiana Ciciriello, Suzana M. Anjos, Annamaria Carissimo, Jie Liao, Graeme W. Carlile, Haouaria Balghi, Renaud Robert, Alberto Luini, John W. Hanrahan, David Y. Thomas. Ouabain Mimics Low Temperature Rescue of F508del-CFTR in Cystic Fibrosis Epithelial Cells. Front Pharmacol. 2012; 3: 176. Published online 2012 October 4. doi: 10.3389/fphar.2012.00176