Grp78

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

GRP78 is a protein that plays a critical role in the endoplasmic reticulum (ER) stress response. It serves as a chaperone, assisting in the folding and processing of proteins within the ER. GRP78 is involved in maintaining ER homeostasis and helping to resolve ER stress by regulating the unfolded protein response.

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Anti-GRP78 (C-20; GRP78 antibody Sh-GRP78 Lentivirus GRP78 siRNA Mouse anti-GRP78 monoclonal antibody Primary antibodies against GRP78 GRP78-N20 (#sc-1050) and DCD-N20 (#sc-27465) antibodies GRP78 (H-129, Anti-Grp78 goat polyclonal Rabbit anti-GRP78

102 protocols using grp78

Immunofluorescence Analysis of Stomach Tissue

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Paraffin sections from animals’ stomachs were deparaffinised and hydrated to distilled water. Sections were then blocked with 5% BSA solution in PBS for 1 hour at room temperature and then incubated with the appropriate primary antibody overnight at 4 °C. The primary antibody used was the anti-human Transthyretin (TTR) from DAKO (A000202) at a dilution of 1/500. The slides were then incubated with Invitrogen Alexa Fluor 555 fluorescence secondary antibody for 1hour at room temperature at 1/2,000 (anti-rabbit A-21428). Finally sections were washed with PBS and mounted using the DAKO Fluorescence Mounting Medium (S3023).
Further analysis was carried out using supplementary antibodies; Fas (Santa Cruz Biotechnology anti-rabbit sc-10231/1000) and activated Caspase-3 (Santa Cruz Biotechnology anti-goat sc-1225 1/500) to assess apoptosis, GRP78 (Santa Cruz Biotechnology anti-rabbit sc-13968 1/1000) to assess the endoplasmic reticulum stress response, and C5b-9 (EMD Millipore anti-rabbit 204903 1/4,000) to assess complement activation and Megalin (Santa Cruz Biotechnology anti-goat sc-16478 1/400). The appropriate secondary antibodies were used, anti-rabbit (Alexa Fluor A-21428 1/2000) and anti-goat (Alexa Fluor A-21432 1/2000). ECL was used to visualize the antibodies as previously explained.

Panayiotou E., Papacharalambous R., Antoniou A., Christophides G., Papageorgiou L., Fella E., Malas S, & Kyriakides T. (2016). Genetic background modifies amyloidosis in a mouse model of ATTR neuropathy. Biochemistry and Biophysics Reports, 8, 48-54.

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Analyzing Cardiac Protein Expression

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Western blotting was used to analyze protein expression. Samples were obtained from left ventricle myocardial tissue, and protein concentration was assayed by a bicinchoninic acid protein assay kit (Pierce, USA). Total protein (50 μg per sample) was loaded on 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes. Following blocking with 5% nonfat milk, the PVDF membranes were incubated with primary antibodies against nerve growth factor (NGF; Abcam, Cat. No. ab52918, 1 : 1000), thyroid hormone (TH; Abcam, Cat. No. ab137869, 1 : 5000), growth associated protein 43 (GAP43; Abcam, Cat. No. ab16053, 1 : 1000), 78-kDa glucose-regulated protein (GRP 78; Santa Cruz, Cat. No. sc-376768, 1 : 1000), C/EBP homologous protein (CHOP; Cell Signaling Technology, Cat. No. 2895, 1 : 1000) at 4°C overnight, followed by secondary antibody incubated for 1.5 h at room temperature. Images were developed using an enhanced chemiluminescence substrate (ECL Plus, GE Healthcare, United States) for 1 min in the dark. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH; ABclonal, Cat. No. AC001, 1 : 1000) was used as an internal loading control.

Wang Q., Chen R., Tang Q., Chen J.Y., Wang Y., Sui D.J, & Liu A.D. (2022). Flavonoid Extract from Propolis Provides Cardioprotection following Myocardial Infarction by Activating PPAR-γ. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 1333545.

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Immunoblotting Analysis of Cellular Proteins

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Rabbit antibodies against GADD153, GRP78, Beclin-1, and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), while those against LC-3 were purchased from MBL. Secondary horseradish peroxidase- (HRP-) conjugated goat anti-rabbit and goat anti-mouse antibodies were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Cells were lysed in a buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 0.1 mM phenylmethylsulfonyl fluoride, 8 μg/ml aprotinin, and 2 μg/ml leupeptin (pH 7.4). For immunoblotting, proteins resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA), which were then exposed to primary and then secondary antibodies. Chemiluminescence detection was performed with an ECL™ Western Blotting Detection Kit (Amersham). The signal intensities of the corresponding bands were measured by a Light Capture equipped with CS Analyzer software (ATTO, Osaka, Japan).

Hirasawa M, & Kurita-Ochiai T. (2018). Porphyromonas gingivalis Induces Apoptosis and Autophagy via ER Stress in Human Umbilical Vein Endothelial Cells. Mediators of Inflammation, 2018, 1967506.

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Histological Assessment of Hepatic Fibrosis

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Haemotoxylin and Eosin, Masson’s Trichrome and Sirius Red (Sigma-Aldrich, United States) stains were completed on 10% Buffered formalin fixed paraffin sections. Collagen content of the liver was quantified histologically using computerised quantification of picroSirius Red staining, as described previously¹⁵. Oil-Red-O (Sigma-Aldrich, USA) staining was performed on frozen OCT embedded cryosections as previously described^{38 (link)}. All sections were visualized on a slide scanner (Virtual Slide System VS120, Olympus, Tokyo, Japan) and viewed in the supplied program (OlyVIA Build 10555, Olympus, Tokyo, Japan). Briefly, for immunofluorescence staining, 4% PFA fixed frozen liver sections were dual stained with both anti-CML (1:125 dilution; ab30917; Abcam, United Kingdom) and OST48 (1:100 dilution; sc25558; Santa Cruz biotechnologies, United States). Dual staining on paraffin buffered formalin fixed sections was with GRP78 (1:50 dilution; sc1050; Santa Cruz biotechnologies, United States) and OST-48 (1:100 dilution; sc74407; Santa Cruz biotechnologies, United States).

Zhuang A., Yap F.Y., Bruce C., Leung C., Plan M.R., Sullivan M.A., Herath C., McCarthy D., Sourris K.C., Kantharidis P., Coughlan M.T., Febbraio M.A., Hodson M.P., Watt M.J., Angus P., Schulz B.L, & Forbes J.M. (2017). Increased liver AGEs induce hepatic injury mediated through an OST48 pathway. Scientific Reports, 7, 12292.

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Naclynamide Synthesis and Antibody Production

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Unless otherwise specified general laboratory reagents and chemicals were provided by ThermoFisher Scientific, Inc.
The signaling peptide, acALY18, was synthesized as previously described.⁹ (link) The active pharmaceutical ingredient (Naclynamide) was synthesized in accordance with the US FDA cGMP standards by the contract manufacturer, Peptisyntha, Inc., a division of Solvay USA. The methodology was that as previously published with the modification as a C-terminal amide.
The acALY18 polyclonal antibody was prepared for TherimuneX Pharmaceuticals Inc., by Lampire Biological Laboratories. GRP78, GAPDH, and XBP1 antibodies were purchased from Santa Cruz Biotechnology. Primary human fibroblasts (accession number GM04190) were purchased from Coriell Institute for Medical Research and were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin.

Overley-Adamson B., Artlett C.M., Stephens C., Sassi-Gaha S., Weis R.D, & Thacker J.D. (2014). Targeting the unfolded protein response, XBP1, and the NLRP3 inflammasome in fibrosis and cancer. Cancer Biology & Therapy, 15(4), 452-462.

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Western Blot Analysis of ER Stress Markers

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Cells were lysed with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Fisher Scientific, Waltham, MA, USA). Proteins transferred to nitrocellulose membranes were probed with the indicated antibodies against: Calnexin (Millipore), GRP78 (Santa Cruz) and β-actin (Santa Cruz). Primary antibodies were followed by the appropriate HRP-conjugated secondary antibodies (Sigma-Aldrich), and chemiluminescent western blotting substrate (Fisher, Waltham, MA, USA; GE Healthcare, Pittsburgh, PA, USA). The blots were then processed on Bio-Rad ChemiDoc Touch Imaging System (Bio-Rad).

Chandra G., Defour A., Mamchoui K., Pandey K., Mishra S., Mouly V., Sreetama S., Mahad Ahmad M., Mahjneh I., Morizono H., Pattabiraman N., Menon A.K, & Jaiswal J.K. (2019). Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells. Cell Death Discovery, 5, 118.

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Ovarian Cancer Cell Line Transfection

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A2780 and OVCAR-3 human ovarian cancer cell lines were transfected with double-stranded siRNAs (30 nmol/mL), such as GRP78 (Santacruz, Dallas, TX, USA), PERK (Santacruz), CHOP (Bioneer, Oakland, CA, USA), and Nox4 (Santacruz), for 24 h using Lipofectamine 2000 reagent (Invitrogen) in a 6-well plate according to the manufacturer’s protocol.

Kim T, & Ko S.G. (2021). JI017, a Complex Herbal Medication, Induces Apoptosis via the Nox4–PERK–CHOP Axis in Ovarian Cancer Cells. International Journal of Molecular Sciences, 22(22), 12264.

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Western Blot Analysis of Melanoma Cells

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Melanoma cells were lysed in RIPA buffer containing 50 mmol/l Tris–HCl pH 8.0, 150 mmol/l NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% SDS supplemented with freshly added protease and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were diluted in 2 × Laemmli buffer and protein samples (15 μg) were loaded on standard 7% SDS–polyacrylamide gel. After electrophoresis, the proteins were transferred onto Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA) followed by incubation in a blocking solution: 5% nonfat milk in PBS-Tween 0.05% or 5% phospho-BLOCKER (Cell Biolabs, San Diego, CA, USA) in PBS-Tween 0.05%. Primary antibodies detecting PARP, ATF6, IRE1α, GRP78 and p53 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), p-IRE1α from Abcam (Cambridge, UK), β-actin from Sigma-Aldrich, p-ERK1/2 (Thr²⁰²/Tyr²⁰⁴) and ERK1/2 from Cell Signaling Technology (Danvers, MA, USA). Secondary HRP-conjugated anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology) and Pierce ECL Western Blotting Substrate (Pierce, Rockford, IL, USA) were used to visualize proteins on the X-ray film (Foton-Bis, Bydgoszcz, Poland) or by using ChemiDoc Imaging System (Biorad). The quantification of the Western blotting data was performed by using ImageJ software.

Mielczarek-Lewandowska A., Sztiller-Sikorska M., Osrodek M., Czyz M, & Hartman M.L. (2019). 17-Aminogeldanamycin selectively diminishes IRE1α-XBP1s pathway activity and cooperatively induces apoptosis with MEK1/2 and BRAFV600E inhibitors in melanoma cells of different genetic subtypes. Apoptosis, 24(7), 596-611.

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Antibody Immunodetection Protocol

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The following primary antibodies were used in this study: BrdU (Biorad, MCA2060 or Abcam, ab6326, 1:200 dilution); DNA (Progen, AC-30-10, 1:250 dilution); GAPDH (Sigma, G8795 or Abcam, ab8245, 1:10,000 and 1:2000 dilutions, respectively); GRP78 (Santa Cruz Biotech, Sc-13968, 1:1000 dilution); HSP60 (Abcam, ab46798, 1:1000 dilution); LC3B (Sigma, L7543) 1:5000; MTCO-2 (Abcam, ab110258 1:1000 dilution); NDUFB8 (Abcam, ab110242, 1:1000 dilution); AMPK alpha (Cell Signaling, 2532, 1:1000 dilution); phosphoAMPK alpha (Cell Signaling, 2531, 1:1000 dilution); TOM20 (Santa Cruz Biotech or Abcam, Ab186735, 1:4000 and 1:10,000 dilutions, respectively); VCL (Abcam, ab18058, 1:1000 dilution); and proliferating cell nuclear antigen (PCNA) (Mouse, sc-56, 1:8000 dilution).
Secondary Antibodies: Anti-Mouse IgG (H + L), HRP Conjugate (Promega, W4021, 1:4000 dilution); anti-Rabbit IgG (H + L), HRP Conjugate (Promega, W4011 1:4000 dilution); Alexa Fluor^®−488 goat- anti-mouse (Invitrogen, A-10684,1:1000 dilution); Alexa Fluor^®−568 goat- anti-mouse (Invitrogen, A-11004, 1:1000 dilution); Alexa Fluor^® 568 donkey anti-rabbit (Invitrogen, A-10042, 1:1000 dilution); Alexa Fluor^®-488 goat- anti-rat (Invitrogen, A-11006, 1:1000 dilution).

Pantic B., Ives D., Mennuni M., Perez-Rodriguez D., Fernandez-Pelayo U., Lopez de Arbina A., Muñoz-Oreja M., Villar-Fernandez M., Dang T.M., Vergani L., Johnston I.G., Pitceathly R.D., McFarland R., Hanna M.G., Taylor R.W., Holt I.J, & Spinazzola A. (2021). 2-Deoxy-D-glucose couples mitochondrial DNA replication with mitochondrial fitness and promotes the selection of wild-type over mutant mitochondrial DNA. Nature Communications, 12, 6997.

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Antibodies and Reagents for ER Stress Study

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Primary antibodies targeting the following proteins were used in this study: p-eIF2α, IRE-1α, CHOP (Cell Signaling Technologies, Danvers, MA, USA), GRP78, ATF6α, PERK, p-PERK, sXBP-1, β-actin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), p-IRE-1α (Abcam, Cambridge, MA, USA) and monoclonal PDI (clone 1D3, Enzo Life Sciences, Farmingdale, NY, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology. IC87114 was obtained from Calbiochem (San Diego, CA, USA). Ovalbumin, lipopolysaccharide, N-ethylmaleimide (NEM), 4-hydroxy-2-nonenal (4-HNE) and dihydroethidine (DHE) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Kim H.K., Lee G.H., Bhattarai K.R., Junjappa R.P., Lee H.Y., Handigund M., Marahatta A., Bhandary B., Baek I.H., Pyo J.S., Kim H.K., Chai O.H., Kim H.R., Lee Y.C, & Chae H.J. (2018). PI3Kδ contributes to ER stress-associated asthma through ER-redox disturbances: the involvement of the RIDD–RIG-I–NF-κB axis. Experimental & Molecular Medicine, 50(2), e444-.

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