Anti hsc70

Manufactured by Abcam

Sourced in United States, United Kingdom

Anti-Hsc70 is a laboratory antibody product used to detect the Hsc70 protein. Hsc70 is a constitutively expressed heat shock protein involved in protein folding and transport. The antibody can be used in various immunological techniques to identify and quantify Hsc70 expression.

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Lab products found in correlation

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Spelling variants of this product

HSC70 (15 citations) Anti-HSC70 antibody (2 citations) Anti-rabbit HSC70 antibody (1 citations)

10 protocols using anti hsc70

Protein Extraction and Western Blot Analysis

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To extract proteins, sciatic nerves were lysed using a RIPA buffer containing 0.1% sodium dodecyl sulfate, 0.5% sodium deoxylate, 150 mM sodium chloride, 100 mM EDTA, 50 mM Tris-hydrochloride, 1% Tergitol, 1% of the protease and phosphatase inhibitors and 1 mM phenylmethylsulfonyl fluoride. The lysates were left to rotate overnight at 4 °C. The following day, lysates were sonicated for 10 cycles 30s/cycle at 4 °C and then centrifuged at 13,600 rpm for 30 min at 4 °C. Protein concentration was measured using the Lowry Assay.
For immunoblotting, 20–40 μg of proteins were separated on 15% polyacrylamide gel electrophoresis (Bio-Rad, CA, USA) and transferred to nitrocellulose membranes (Bio-Rad). The blots were blocked with 5% BSA (Sigma-Aldrich, Darmstadt, Germany) in Tris-buffered saline for 1 h and then incubated overnight with mouse polyclonal anti-CYP 1a1/1a2 (1:500, Detroit R&D, Michigan, USA), and mouse polyclonal anti-HSC70 (1:1000, Abcam). The primary antibodies were detected using horseradish peroxidase–conjugated IgG (1:10000, Bio-Rad). Bands were visualized by the stain-free chemidoc imaging system (Bio-Rad). Densitometric analysis was performed using Image J software.

Dannawi M., Riachi M.E., Haddad A.F., El Massry M., Haddad M., Moukarzel P., Harb F., Ghadieh H.E, & Eid A.A. (2022). Influence of intermittent fasting on prediabetes-induced neuropathy: Insights on a novel mechanistic pathway. Metabolism Open, 14, 100175.

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Protein Profiling of Cells and Exosomes

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Cells and exosomes were lysed using CHAPS lysis buffer (30 mM Tris-Cl, pH 7.5; 150 mM NaCl; 1% CHAPS) mixed with 25X protease inhibitor (Roche), and sonication. Proteins were quantified by Bradford method (Bio-Rad). Electrophoresis was performed using 10% acrylamide gel, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and probed with the following antibodies: anti-TSG101 (Abcam), anti-HSC70, anti-GAPDH, anti-Calnexin, anti-C/EBPβ, anti-CDK2 (all from Santa Cruz Biotechnology), anti-FOXO-1 (Cell Signaling), and appropriate species-specific HRP-conjugated secondary antibodies (Santa Cruz), and detected using ECL reagent (Roche).

Ghayad S.E., Rammal G., Ghamloush F., Basma H., Nasr R., Diab-Assaf M., Chelala C, & Saab R. (2016). Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts. Scientific Reports, 6, 37088.

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Immunofluorescence and Immunohistochemistry for GKN2 Analysis

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Cells were seeded in 6-well plates, incubated for 24 h and treated with H₂O₂ or phosphate-buffered saline (PBS) for 6 h. The primary antibodies used were anti-GKN2 and anti-Hsc70 (Abcam). 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, China) was used for nuclear staining. Alexa Fluor® 488 anti-rabbit and Alexa Fluor® 594 anti-mouse antibodies (CST) were used as secondary antibodies. Immunofluorescence assays were performed as previously described [22 (link)]. Tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. anti-GKN2 (1:200; Abcam) and anti-Ki67 (1:200; CST) were used as primary antibodies. Immunohistochemistry was performed as previously described [23 (link)]. GC tissues and adjacent non-malignant gastric tissues were obtained from the Huashan Hospital (Shanghai, China). The use of all tissue samples was approved by the Institutional Ethics Review Board of the Huashan Hospital.

Zhang Z., Xue H., Dong Y., Zhang J., Pan Y., Shi L., Xiong P., Zhu J., Li W., Zheng W., Liu J, & Du J. (2019). GKN2 promotes oxidative stress-induced gastric cancer cell apoptosis via the Hsc70 pathway. Journal of Experimental & Clinical Cancer Research : CR, 38, 338.

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Western Blot Analysis of Acetylation

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CD41⁺ cells were harvested at different times of culture, washed once in 1× PBS, and lysed in Nonidet P40 (NP40) buffer containing protease and phosphatase inhibitors (Roche)^{14 (link)}. Lysates were gently sonicated on ice and protein concentration was assessed using Bradford assay (Bio-Rad). Protein samples were subjected to SDS–polyacrylamide gel electroporesis. After transfer, nitrocellulose membranes were blotted with the following 1/1000-diluted antibodies: anti-HDAC6, anti-acetylated cortactin, and anti-cortactin (from Millipore, catalog number 07-732, 09-881, and AB3887), anti-ac-tubulin, anti-tubulin, and anti-actin (Sigma, catalog number T7451, T9026 clone DM1A, and A2228 clone AC-74), anti-acetylated histone H3 (Cell Signaling, catalog number 4353), and anti-HSC70 (Abcam, ab19136), followed by revelation by horseradish peroxidase-linked secondary antibodies and Chemiluminescent HRP Substrate (Millipore) using IQuant Instrument (GE Healthcare).

Messaoudi K., Ali A., Ishaq R., Palazzo A., Sliwa D., Bluteau O., Souquère S., Muller D., Diop K.M., Rameau P., Lapierre V., Marolleau J.P., Matthias P., Godin I., Pierron G., Thomas S.G., Watson S.P., Droin N., Vainchenker W., Plo I., Raslova H, & Debili N. (2017). Critical role of the HDAC6–cortactin axis in human megakaryocyte maturation leading to a proplatelet-formation defect. Nature Communications, 8, 1786.

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Western Blot Analysis of Protein Expression

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Protein extracts were prepared by lysing cells in RIPA buffer. Protein yield was quantified using the Bio-Rad DC protein assay kit (Bio-Rad, USA). Equal amounts of protein were separated by 10% SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) before wet-transfer onto PVDF membrane (Amersham Biosciences, UK). Nonspecific binding sites were blocked by overnight incubation with 5% nonfat dry milk in Tris-buffered saline with 1% Tween (TBS-T; 130 mmol/L NaCl, 20 mmol/L Tris, pH7.6 and 1% Tween). Primary antibodies used were anti-HSC70 (Abcam, UK), anti-BiP, anti-Calnexin, anti-ERo1α, anti-CHOP, anti-PERK, anti-PDI, anti-LC3B, anti-COX2 (Cell Signaling, USA) and β-ACTIN (Abcam, UK) which was used as a loading control. All primary antibodies were diluted 1:1000; except for the anti-β-ACTIN, which was diluted 1:100,000. Full length scans are presented as supplementary information. Note that some images were reflected for consistency in the sequence of presentation.

Brosens J.J., Salker M.S., Teklenburg G., Nautiyal J., Salter S., Lucas E.S., Steel J.H., Christian M., Chan Y.W., Boomsma C.M., Moore J.D., Hartshorne G.M., Šućurović S., Mulac-Jericevic B., Heijnen C.J., Quenby S., Groot Koerkamp M.J., Holstege F.C., Shmygol A, & Macklon N.S. (2014). Uterine Selection of Human Embryos at Implantation. Scientific Reports, 4, 3894.

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Western Blot Analysis of Neurodegenerative Markers

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After determination of protein content using a bicinchoninic acid (BCA) assay (Pierce Biotechnology, Waltham, MA, USA), cell extracts containing 15 µg of total protein were separated on 4–15% precast midi protein gels (Bio-Rad, Hercules, CA, USA) and electroblotted onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were first incubated with 0.4% paraformaldehyde (PFA) for 15 min for better αsyn binding, then blocked with PBST with 5% milk for 30 min and incubated with the following primary antibodies: anti–α-Syn (BD Transduction, San Jose, CA, USA), anti-α-tubulin (Sigma-Aldrich, Saint Louis, MO, USA), anti-ubiquitin (Abcam, Cambridge, MA, USA), anti-GAPDH (Abcam, Cambridge, MA, USA), anti-β-catenin (Abcam, Cambridge, MA, USA), anti-Hsc70 (Abcam, Cambridge, MA, USA), anti-Hsp90 (Abcam, Cambridge, MA, USA), anti-LC3 (Novus Biologicals, Centennial, CO, USA), and anti-ATP13A2 (Sigma-Aldrich, Saint Louis, MO, USA). After overnight incubation with primary antibodies at 4 °C, blots were washed three times with PBS/1%Triton and incubated with horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) for 1 h. Detection was performed using chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) and an LAS-4000 imaging system (Fujifilm, Saitama, Japan).

Si J., Van den Haute C., Lobbestael E., Martin S., van Veen S., Vangheluwe P, & Baekelandt V. (2021). ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization. International Journal of Molecular Sciences, 22(5), 2689.

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Characterization of Protein Signaling Pathways

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Cell lysates were extracted with a cell lysis buffer (Beyotime, Hangzhou, China) and the protein concentration was quantified using an Enhanced BCA Protein Assay Kit (Beyotime). The primary antibodies used were as follows: anti-p65 (1:1000), anti- phosphorylated p65 (1:1000), anti-JNK (1:1000), anti-phosphorylated JNK (1:1000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000), anti-cleaved caspase-3 (1:1000), anti-cleaved caspase-9 (1:1000), anti-cleaved PARP (1:1000) (CST, Danvers, MA, USA); anti-GKN2 (1:1000), anti-Hsc70 (1:1000) (Abcam, Cambridge, MA, USA). Anti-rabbit antibody (1:2000) and anti-mouse antibody antibodies (1:2000) (CST) were used as secondary antibodies. Western blot was performed as previously described [21 (link)].

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Antibody Sourcing for Cell Biology

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Thapsigargin and BFA were purchased from Sigma-Aldrich (St Louis, MO, USA). Apoptozole was purchased from Millipore (Billerica, MA, USA). Anti-pendrin (G-19, epitope against C terminus), anti-HA, anti-Myc, anti-aldolase and anti-Flag antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-pendrin (ab66702, epitope against N terminus), anti-calnexin, anti-giantin, anti-Lamp1, anti-CHMP2B, anti-ATG7 and anti-Hsc70 were from Abcam (Cambridge, UK); anti-EEA1 was from BD Biosciences; anti-CD9 was from LSBio (Seattle, WA, USA); anti-DNAJC14 was from Novus (Littleton, CO, USA); anti-Rab7 was from Cell Signaling Technologies (Danvers, MA, USA); and anti-CFTR antibody (M3A7) was from Millipore. Detailed information for primary and secondary antibodies used in this study is summarized in Supplementary Table 4.

Jung J., Kim J., Roh S.H., Jun I., Sampson R.D., Gee H.Y., Choi J.Y, & Lee M.G. (2016). The HSP70 co-chaperone DNAJC14 targets misfolded pendrin for unconventional protein secretion. Nature Communications, 7, 11386.

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Immunoblotting Techniques for Protein Analysis

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The following antibodies were used: anti-TG2 (Thermo Scientific, CUB7402), anti-PKM2 (Cell Signalling, D78A4), anti-EEF1A1 (Milipore, 05-235), anti-Hsc70 (Abcam, 51052), HSPA1A (Santa Cruz, 7947), anti-Biotin (Abcam, 1227), anti-p62/SQSTM1 (mbl, PM045), anti-LC3 (Novus Biologicals, NB100-2331), anti-GAPDH (Sigma, G9545), anti-Actin (Sigma, 2066), anti-Beclin1 (Santa Cruz, 10086), anti-HIF1-beta (Novus Biologicals, NB100-124), anti-ULK1(Santa Cruz, 33182), anti-phospho-tyrosine (Cell Signaling, 9411) and HRP-conjugated secondary antibodies (Bio-Rad Laboratories).

Altuntas S., Rossin F., Marsella C., D'Eletto M., Hidalgo L.D., Farrace M.G., Campanella M., Antonioli M., Fimia G.M, & Piacentini M. (2015). The transglutaminase type 2 and pyruvate kinase isoenzyme M2 interplay in autophagy regulation. Oncotarget, 6(42), 44941-44954.

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Western Blot Analysis of Autophagy Markers

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After treating under various conditions, the tissues or cells were harvested, the total proteins were extracted, and the total protein concentrations were detected by BCA Protein Assay Kit (Beyotime Biotechnology, Nantong, China). Equivalent amounts of protein of each sample were electrophoresed on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After the membranes were blocked for 1 h at RT, primary antibodies (in TBST-5% BSA) against SNCA, LAMP-2A, Hsc70, LC3, Beclin-1, P62, and Nrf2 (anti-SNCA, anti-LAMP-2A, anti-Hsc70, and anti-Nrf2 antibodies are obtained from Abcam, MA, USA; anti-LC3, anti-Beclin-1, and anti-P62 antibodies are obtained from Cell Signaling Technology, Beverly, USA) were added and incubated overnight at 4°C. After being washed 3 times in TBST, the membranes were incubated with HRP-conjugated secondary antibodies (KPL, Gaithersburg, MD, USA) for 1 h at 37°C. An ECL kit (Millipore, Bedford, MA, USA) was used to visualize membrane immunoreactivity.

Zhang J.X., Tong W.F., Jiang M., Zhou K.G., Xiang X.R., He Y.J., Zhang Z.Y., Guan Q, & Jin L.J. (2022). MANF Inhibits α-Synuclein Accumulation through Activation of Autophagic Pathways. Oxidative Medicine and Cellular Longevity, 2022, 7925686.

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