Hsp90

Manufactured by Enzo Life Sciences

Sourced in United States

HSP90 is a heat shock protein that functions as a molecular chaperone, facilitating the folding and stabilization of various client proteins. It is a key regulator of protein homeostasis and plays a crucial role in maintaining the structural and functional integrity of numerous cellular proteins.

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Lab products found in correlation

Hsp70, by Enzo Life Sciences (5 mentions) Las4000 imager, by GE Healthcare (4 mentions) Las 4000, by Cytiva (4 mentions) β actin, by Merck Group (3 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Ecl western blotting detection reagent, by GE Healthcare (2 mentions) Immobilon p, by Merck Group (2 mentions) Species specific biotinylated antibodies, by Enzo Life Sciences (2 mentions) Horseradish peroxidase, by Enzo Life Sciences (2 mentions) Flag tag (flag), by Merck Group (2 mentions) Grp94, by Enzo Life Sciences (2 mentions) Gapdh, by Enzo Life Sciences (2 mentions) Ubiquitin, by Santa Cruz Biotechnology (2 mentions) Mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions) Lamin b, by Bioworld Technology (2 mentions) Gapdh, by Bioworld Technology (2 mentions) Tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions) P foxo1, by Cell Signaling Technology (2 mentions) Sirt6, by Cell Signaling Technology (2 mentions) Foxo1, by Cell Signaling Technology (2 mentions)

Spelling variants of this product

Anti-Hsp90 (9 citations) HSP90 mAb (6 citations) Hsp90-835-16F1 (2 citations) Anti-Hsp90 (ADI-SPA-835, (2 citations) Anti-Hsp90 antibody (2 citations) HSP90 antibody (2 citations)

23 protocols using hsp90

Immunoblotting Analysis of Cellular Proteins

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Cells were lysed with 1% Triton X-100 lysis buffer (20 mM Tris-HCl at pH 7.4, 5 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄, 1% Triton X-100) supplemented with protease inhibitor cocktail (Roche). Equal amount of total cellular proteins per sample was subjected to SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad Laboratories). Antibodies for immunoblotting included anti-p53 (DO-1, Santa Cruz Biotechnology), p21 (Cell Signaling), PUMA (Cell Signaling), MDM2 (Calbiochem), Noxa (Calbiochem), Hsp40 (Santa Cruz Biotechnology), Hsp90 (Enzo Life Sciences), and β-actin (Sigma). Bands were detected using Western Lightning Plus ECL (PerkinElmer) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). All experiments were performed independently at a minimum of three times.

Hiraki M., Hwang S.Y., Cao S., Ramadhar T.R., Byun S., Yoon K.W., Lee J.H., Chu K., Gurkar A.U., Kolev V., Zhang J., Namba T., Murphy M.E., Newman D.J., Mandinova A., Clardy J, & Lee S.W. (2015). Small molecule reactivation of mutant p53 to wt-like p53 through the p53-Hsp40 regulatory axis. Chemistry & biology, 22(9), 1206-1216.

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Immunoblot Analysis of Retinal Proteins

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Immunoblot analysis was performed according to a standard protocol. Briefly, after determining protein concentrations (BCA Protein Assay Kit, Pierce, Rockford, IL), 1ug of detergent-soluble retinal proteins was separated on precast 12% SDS-polyacrylamide gels (Mini-Protein TGX, BIO-RAD, Hercules, CA) and transferred onto PVDF membranes (Immobilon-P, Millipore, Billerica, MA). Membranes were blocked with 5% non-fat milk and incubated with primary antibodies for Hsp70 (1:5000; Enzo, Farmingdale, NY), Hsp90 (1:5000; Enzo), Hsf1 (1:1000; Enzo) or Hsf2 (1:1000; Enzo) at 4°C overnight. Membranes were subsequently incubated with species-specific biotinylated antibodies (1:500; Enzo) and Horseradish Peroxidase (1:400; Enzo) for 1 hour at 4°C. Immunoreactive bands were detected with ECL Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ), and their intensities were quantified with NIH image software. Reprobing blots with a monoclonal antibody to a housekeeping beta-actin (1:10000; Sigma, St. Louis, MO) was used as an internal reference to normalize protein expression levels.

Kwong J.M., Gu L., Nassiri N., Bekerman V., Kumar-Singh R., Rhee K.D., Yang X.J., Hauswirth W.W., Caprioli J, & Piri N. (2014). AAV-mediated and pharmacological induction of Hsp70 expression stimulates survival of retinal ganglion cells following axonal injury. Gene therapy, 22(2), 138-145.

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Immunoprecipitation and Western Blot Analysis of Stress Response Proteins

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Cells were lysed with TGNET buffer [50 mM Tris HCl pH7.5, 5% Glycerol, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100] containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). Lysates were centrifuged at maximum speed for 15 minutes at 4 °C. Supernatants were subjected to BCA protein assay for normalization, and then 40 μg of protein were used as input lysate to confirm protein expression. From remaining lysates, 700 μg of protein were subjected to immunoprecipitation by addition of 40 μl of Anti-FLAG M2 affinity beads (Sigma-Aldrich, A2220) or HA affinity beads (clone HA-7, Sigma-Aldrich, A2095) followed by incubation with rotation for 2 hours at 4 °C. After centrifugation, beads were washed 4 times with TGNET buffer. The proteins were then eluted with 30 μl of 2X SDS sample buffer by boiling at 95 °C for 5 minutes. Subsequently, samples were subjected to SDS-PAGE followed by western blotting. The following antibodies were used for western blot: HSF1 (Santa Cruz, sc-13516), HSP90 (Enzo Life Science, ADI-SPA-835), HSP70 (Enzo Life Sciences, ADI-SPA-810), HA (Rockland, 600-401-384), and FLAG (Sigma-Aldrich, A8592).
Additional Methods may be found in Supplemental Information.

Kijima T., Prince T.L., Tigue M.L., Yim K.H., Schwartz H., Beebe K., Lee S., Budzynski M.A., Williams H., Trepel J.B., Sistonen L., Calderwood S, & Neckers L. (2018). HSP90 inhibitors disrupt a transient HSP90-HSF1 interaction and identify a noncanonical model of HSP90-mediated HSF1 regulation. Scientific Reports, 8, 6976.

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Western Blot Analysis of Signaling Proteins

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Cell lysates (20 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% nonfat dry milk, the membrane was probed with primary antibodies against p38, p-p38, p-AKT, AKT, p-ERK, ERK, JNK, p-JNK, p65, p-IκBα, IκBα (Cell Signaling, Beverly, MA, USA), or HSP90 (Enzo Life Sciences, Farmingdale, NY, USA). Membrane was incubated with goat anti-rabbit horseradish peroxidase–conjugated IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The proteins of interest on immunoblots were detected by using Las-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).

Kim S.H., Zhang Z., Moon Y.J., Park I.W., Cho Y.G., Jeon R, & Park B.H. (2018). SPA0355 prevents ovariectomy-induced bone loss in mice. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 23(1), 47-54.

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Platelet Proteome Analysis via Western Blot

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After treatment with the indicated inhibitors with or without
thrombin-mediated activation, platelet whole cell lysates were generated in TNES
buffer (50mM Tris pH7.5, 100mM NaCl, 2mM EDTA, 1% NP-40 supplemented with
protease and phosphatase inhibitors (cOmplete and PhosSTOP cocktail tablets,
respectively, Sigma-Aldrich, St. Louis, MO, USA). After cellular debris was
removed via high speed centrifugation, equal amounts of the lysates were
fractionated on gradient 4–20% SDS-PAGE gels and transferred to
nitrocellulose membranes (both from Bio-Rad, Hercules, CA, USA). The membranes
were analyzed for immunoreactivity to the following antibodies: Hsp40, (Cell
Signaling Technology, Danvers, MA, USA), Hsp70, Hsp90, and Grp94 (Enzo Life
Sciences, Farmingdale, NY, USA), and α-tubulin (EMD Millipore,
Burlington, MA, USA). Bound antibodies were detected via species-specific
horseradish peroxidase (HRP)- conjugated secondary antibodies at a 1:000
dilution (GE Healthcare, Pittsburgh, PA, USA), followed by reaction with Pico
ECL (Thermo Scientific, Rockford, IL, USA) to produce a chemiluminescent signal,
and exposed to x-ray film (Vita Scientific, College Park, MD, USA).

Jackson J.W., Rivera-Marquez G.M., Beebe K., Tran A.D., Trepel J.B., Gestwicki J.E., Blagg B.S., Ohkubo S, & Neckers L.M. (2020). Pharmacologic dissection of the overlapping impact of heat shock protein family members on platelet function. Journal of thrombosis and haemostasis : JTH, 18(5), 1197-1209.

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Immunoblot Analysis of Apoptosis Regulators

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Immunoblots were carried out as described by our group [23 ,24 (link)]. Original immunoblots see File S1. Antibodies used for this assay were: BCL-XL (#ab32370), survivin (#ab134170), p21 (#ab109520), BAX (#ab32503), BAK (#ab32371), BIM (#ab32158), HDAC3 (#ab32369), GAPDH (#ab128915) from Abcam, Cambridge, UK; MCL-1 (#sc-819), HDAC8 (#sc-374180), HSP90 (#sc-13119), vinculin (#sc-73614) from Santa Cruz Biotechnology, Heidelberg, Germany; cleaved caspase-3 (#cs9661), PARP1 (#cs9542), BID (#cs2002), HDAC1 (#cs34589), HDAC2 (#cs5113), histone H3 (#cs14269), ac-histone H3 (K9) (#cs9649), ac-histone H3 (K18) (#cs9675), ac-histone H3 (K27) (#cs8173) from Cell Signaling, Leiden, The Netherlands; ac-tubulin (#T7451) from Sigma-Aldrich, Taufkirchen, Germany; ac-histone H3 (#06-599) from Merck Millipore, Burlington, MA, USA; and NOXA (#ALX-804-408) from Enzo Life Sciences, New York, NY, USA. HSP90, GAPDH, and vinculin served as independent housekeeping proteins to normalize protein loading. The protein ladders used were the PageRuler^TM pre-stained protein ladder (#26616) and the PageRuler^TM Plus pre-stained protein ladder (#26619) from Thermo Fischer Scientific, Waltham, MA, USA.

Ashry R., Mustafa A.H., Hausmann K., Linnebacher M., Strand S., Sippl W., Wirth M, & Krämer O.H. (2023). NOXA Accentuates Apoptosis Induction by a Novel Histone Deacetylase Inhibitor. Cancers, 15(14), 3650.

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Western Blot Analysis of Metabolic Proteins

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The proteins in tissue homogenates (20 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, the blots were probed with primary antibodies against Sirt6, Akt, p-Akt (Ser473), p38 MAPK, p-p38 MAPK, p-ATGL (Ser406), HSL, p-HSL (Ser563, Ser565, Ser660), CREB, p-CREB (Cell Signaling, Beverly, MA, USA), UCP1, ATGL (Abcam), HSP90 (Enzo Life Sciences, Plymouth Meeting, PA, USA), ATF2, and p-ATF2 (Santa Cruz Biochemicals, Dallas, TX, USA). Immunoreactive bands were detected with a Las-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA).

Wu D., Bang I.H., Park B.H, & Bae E.J. (2021). Loss of Sirt6 in adipocytes impairs the ability of adipose tissue to adapt to intermittent fasting. Experimental & Molecular Medicine, 53(9), 1298-1306.

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Immunoprecipitation and Immunoblotting of VHL-HIF1α Complexes

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Proteins were extracted from transiently transfected human embryonic kidney (HEK293) as previously described. (Woodford et al. 2016 (link)) For immunoprecipitation, cell lysates were incubated with anti-FLAG antibody conjugated beads (Sigma) for 2 h at 4ºC. Immunopellets were washed 3 times with lysis buffer (20 mM HEPES (pH7.0), 100-mM NaCl, 1-mM MgCl₂, 0.1% NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)). Precipitated proteins were resuspended in 5X Laemmli buffer, boiled, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Co-immunoprecipitated proteins were detected by immunoblotting. Immunoblotting was performed with the indicated antibodies recognizing VHL, HA, HIF1α, human Mps1 (TTK) (Cell Signaling), Ubiquitin (Santa Cruz Biotechnology), Hsp90, GAPDH (ENZO Life Sciences), 6x-His, and FLAG (Thermo Fisher Scientific).

Woodford M.R., Backe S.J., Wengert L.A., Dunn D.M., Bourboulia D, & Mollapour M. (2021). Hsp90 chaperone code and the tumor suppressor VHL cooperatively regulate the mitotic checkpoint. Cell Stress & Chaperones, 26(6), 965-971.

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Protein Expression and Localization

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Tissues and cells were homogenized in Tissue Protein Extraction Reagent or Mammalian Protein Extraction Reagent (Thermo). Nuclear and cytoplasmic components were isolated using NE-PER nuclear and cytoplasmic extraction kit (Thermo). Homogenates (20 μg of total protein) were separated by SDS-PAGE and transferred to nitrocellulose membranes.
Antibodies were used against the following proteins: Sirt6, Sirt1, Ac-H3K9, FoxO1, p-FoxO1 (Cell Signaling, Beverly, MA, USA), Ac-lysine, Sirt2, Sirt3 (Abcam, Cambridge, UK), ubiquitin (Santa Cruz Biochemicals, Dallas, TX, USA), Sirt4, lamin B, GAPDH (Bioworld Technology, St Louis Park, MN, USA), Sirt5, Sirt7, Ac-FoxO1 (LifeSpan Biosciences, Seattle, WA, USA), and HSP90 (Enzo Life Sciences, Plymouth Meeting, PA, USA).

Shi M.Y., Bang I.H., Han C.Y., Lee D.H., Park B.H, & Bae E.J. (2020). Statin suppresses sirtuin 6 through miR-495, increasing FoxO1-dependent hepatic gluconeogenesis. Theranostics, 10(25), 11416-11427.

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Immunoblot Analysis of Retinal Proteins

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