Adi spa 800

Manufactured by Enzo Life Sciences

Sourced in Belgium, Germany

The ADI-SPA-800 is a piece of laboratory equipment designed for sample preparation. It is a programmable automated liquid handling system capable of performing tasks such as serial dilutions, plate filling, reagent transfer, and sample aliquoting.

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Lab products found in correlation

Adi spa 803, by Enzo Life Sciences (2 mentions) Adi spp 715, by Enzo Life Sciences (1 mentions) Fetal calf serum (fcs), by Harvard Bioscience (1 mentions) Spa 810, by Enzo Life Sciences (1 mentions) Spa 812, by Enzo Life Sciences (1 mentions) Bovine serum albumin (bsa), by Roche (1 mentions) Ab76076, by Abcam (1 mentions) Adi spa 901, by Enzo Life Sciences (1 mentions) Ecl detection system, by GE Healthcare (1 mentions) Adi spa 810, by Enzo Life Sciences (1 mentions) β actin, by Merck Group (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Chemidoc touch imaging system, by Bio-Rad (1 mentions) Mouse monoclonal anti β actin sc 47778, by Santa Cruz Biotechnology (1 mentions) Rabbit polyclonal anti cleaved caspase 3 9661, by Cell Signaling Technology (1 mentions) Rabbit polyclonal anti parp, by Cell Signaling Technology (1 mentions) Mouse monoclonal anti cytochrome c, by BD (1 mentions) Adi spa 400, by Enzo Life Sciences (1 mentions) Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)

Close spelling variants of this product

SPA-800

5 protocols using adi spa 800

Quantifying Cytosolic and Cytoskeletal HSP27

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HSP27 in the cytosolic and cytoskeletal fractions was measured as previously described in detail [5 (link)]. Briefly, HSP27 was detected using a in house-made double antibody sandwich ELISA. By using capture antibodies (25 ng/well; ADI-SPA-800, Enzo Life Sciences) and detection antibodies against HSP27 (ADI-SPA-803, Enzo Life Sciences), HSP27 was determined by using a filter photometer (Expert 96, ASYS Hitech) measuring optical density at 450 nm.

Cumming K.T., Raastad T., Sørstrøm A., Paronetto M.P., Mercatelli N., Ugelstad I., Caporossi D, & Paulsen G. (2017). Vitamin C and E supplementation does not affect heat shock proteins or endogenous antioxidants in trained skeletal muscles during 12 weeks of strength training. BMC nutrition, 3, 70.

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Antibody and protein detection protocol

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HSP70 monoclonal antibody (SPA-810), HSP27 monoclonal antibody (ADI SPA-800), human recombinant HSP70 protein (SPP-755), polyclonal HSP70 antibody (SPA-812), recombinant human HSP27 (ADI-SPP-715), and polyclonal HSP27 antibody (ADI- SPA-803), were from Enzo Lifesciences (Antwerp, Belgium). Ficoll was from Nycomed (Oslo, Norway), Bovine serum albumin (BSA) was from Roche (Boehringer Mannheim, Germany), Fetal calf serum was from Biochrom (International Medical, Wavre, Belgium), RIPA buffer was made up of 1% nonidet P40, 1% deoxycholic acid, NaCl 150 mM, 0.1% SDS, 1% Triton X-100, phosphate inhibitors (50 mM NaF, 10 mM h-glycerophosphate, 1 mM Na₂P₂O₇ 10H₂O, 10 mM Na₃VO₄, 10 mM p-nitrophenylphosphate) and a cocktail of protease inhibitors (10:1000). Fix and Perm cell permeabilization kit was from IMTEC (Antwerp, Belgium). Phosphate-buffered saline (PBS) consisted of 40 mM Na 2HPO₄ 2H₂O, 135 mM NaCl, 3 mM KCl, and 1 mM KH₂PO₄.

Njemini R., Verhaeghen K., Mets T., Weets I, & Bautmans I. (2020). A Novel Bead-Based Immunoassay for the Measurement of Heat Shock Proteins 27 and 70. Pathogens, 9(11), 863.

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Western Blot and ELISA Characterization

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Cells were lysed in TBST buffer as described previously [30 (link)]. The protein content was determined using the BCA™ Protein Assay Kit (Pierce). After transfer on PVDF membranes (BioRad) blots were blocked in 5% skim milk and incubated with antibodies (4 °C, overnight) directed against HSF-1 (ADI-SPA-901; Enzo Life Sciences Inc., Farmingdale, NY, USA), HSF-1 phospho S326 (pHSF-1) (ab76076; abcam), Hsp70 (ADI-SPA-810; Enzo Life Sciences; cmHsp70.1; multimmune GmbH), Hsp27 (ADI-SPA-800; Enzo Life Sciences), Akt (Cell Signaling Technology Europe, Frankfurt, Germany) and β-actin (A5316; Sigma-Aldrich, St. Louis, MO, USA). Horseradish-peroxidase (HRP)-conjugated anti-rabbit/anti-mouse antibodies (Promega, Fitchburg, WI, USA, W401B/W402B) were used as secondary antibodies. Immune complexes as detected by ECL detection system (GE Healthcare, Chicago, IL, USA) were imaged digitally (ChemiDoc Touch Imaging System, Biorad, Hercules, CA, USA). Hsp70 protein concentrations in cell lysates were quantified by ELISA (R&D systems, Minneaplis, MN, USA) following the manufacturer’s recommendations.

Kühnel A., Schilling D., Combs S.E., Haller B., Schwab M, & Multhoff G. (2019). Radiosensitization of HSF-1 Knockdown Lung Cancer Cells by Low Concentrations of Hsp90 Inhibitor NVP-AUY922. Cells, 8(10), 1166.

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Cloning and Characterization of HSP25 Mutants

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Wildtype (WT) mouse HSP25 (GenBank accession number, NM_013560.2) was polymerase chain reaction (PCR)-amplified, and the dimerization mutant HSP25 (C141A) was constructed by overlap extension PCR. The PCR fragments were digested with BamHI-EcoRI and ligated into p3x-Flag-Myc-CMV-26, yielding p3x-flag-HSP25 (WT) and p3x-flag-HSP25 (C141A). WT and dimerization mutant (C141A) HSP25 were cloned into p3x-Flag-myc-cmv-26 expression vector [21 (link)]. Goat polyclonal anti-HSP27 (sc-1049), goat polyclonal anti-HSP70 (sc-1060), mouse monoclonal anti-HSP90α/β (sc-13119), mouse monoclonal anti-HSF1 (sc-17757), rabbit-polyclonal anti-PKCδ (sc-213) and mouse monoclonal anti-β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-cleaved caspase-3 (#9661), rabbit polyclonal anti-cleaved PARP (#9541), and rabbit polyclonal anti-cleaved cytochrome c (#9661) antibodies were purchased from Cell Signaling (Beverly, MA, USA). Mouse monoclonal anti-cytochrome c (#556432, BD Biosciences) and rabbit polyclonal anti-PARP (#9542, Cell signaling) antibodies were used for immuneprecipitation. Mouse-monoclonal anti-HSP27 (ADI-SPA-800, Enzo) antibody was also used for immunohistochemistry.

Kim J.H., Jung Y.J., Choi B., Lee N.L., Lee H.J., Kwak S.Y., Kwon Y., Na Y, & Lee Y.S. (2016). Overcoming HSP27-mediated resistance by altered dimerization of HSP27 using small molecules. Oncotarget, 7(33), 53178-53190.

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Comprehensive Protein Extraction and Western Blot Analysis

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Cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 1% NP40, 250 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 1 mM protease inhibitor (PMSF, aprotinin, leupeptin) and 1 mM DTT. Whole cell lysates were subjected to protein quantification and analyzed by Western blotting. Antibodies used in this study: anti-IER5 rabbit polyclonal antibody (HPA029894), anti-Flag M2 mouse monoclonal antibody (F1804), anti-β-actin mouse monoclonal antibody (A2228) from SIGMA, anti-p53 goat polyclonal antibody (sc-6243-G), anti-p21 rabbit polyclonal antibody (C-19), anti-HSF1 rabbit polyclonal antibody (sc-9144), anti-phospho-HSF1 (Ser230) rabbit polyclonal antibody (sc-30443-R), anti-PP2A-B55 (sc-18330) from Santa Cruz Biotechnology, anti-phospho-HSF1 (Ser320) rabbit monoclonal antibody (#2446-1) and anti-phospho-HSF1 (Ser326) rabbit monoclonal antibody (#2092-1) from Epitomics, anti-phospho HSF1 (Ser121) rabbit polyclonal antibody from Assay bio Tech, anti-phospho-HSF1 (Ser303/Ser307) rabbit monoclonal antibody (ab81281) from abcam, anti-HSPB1 mouse monoclonal antibody (ADI-SPA-800), anti-DNAJB1 rabbit polyclonal antibody (ADI-SPA-400), anti-HSPA6 mouse monoclonal antibody (ADI-SPA-754), anti-HSPA1A/1B mouse monoclonal antibody (ADI-SPA-810) from Enzo Life Sciences, anti-HA mouse monoclonal antibody (12CA5) from Roche, and anti-PP2A C subunit (clone 1D6) from Merck Millipore.

Asano Y., Kawase T., Okabe A., Tsutsumi S., Ichikawa H., Tatebe S., Kitabayashi I., Tashiro F., Namiki H., Kondo T., Semba K., Aburatani H., Taya Y., Nakagama H, & Ohki R. (2016). IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis. Scientific Reports, 6, 19174.

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