Total protein was isolated from the cell cultures following transfection (72 h). Protein lysates were prepared by lysing the cells in ice-cold RIPA buffer (G-Biosciences) supplemented with protease and phosphatase inhibitors (MilliporeSigma), which were diluted 1:10 v/v as per the manufacturer’s recommendations. Cell debris was removed by centrifugation at 16,000 × g at 4°C, and protein concentrations were determined using a Pierce BCA kit (Thermo Fisher Scientific). A sample (20–35 mg) of the supernatant protein was mixed with LDS buffer and DTT, incubated at 70°C for 10 min and resolved on a 4%–12% Bis-Tris PAGE gradient gel before being transferred to a polyvinylidene fluoride (PVDF) membrane. Following transfer, the membrane was blocked in 5% skim milk for 1 h, washed, and incubated at 4°C overnight with a rabbit 1° monoclonal antibody against human GRP78, GRP94, GRP75, or β-actin (all purchased from Cell Signaling Technology) at a 1:1,000 dilution. The membrane was subsequently washed and incubated with an anti-rabbit horseradish peroxidase (HRP)-conjugated 2° Ab (Cell Signaling Technology) for 1 h at room temperature at a 1:2,000 dilution. The bands were visualized using a SignalFire enhanced chemiluminescence (ECL) reagent (Cell Signaling Technology) on a ProteinSimple FluorChem E imager.
Grp75
Manufactured by Cell Signaling Technology
Sourced in United States
GRP75 is a lab equipment product manufactured by Cell Signaling Technology. It is a molecular chaperone that plays a crucial role in the proper folding and transport of proteins within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Proteinsimple fluorchem e imager, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
Pierce bca kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by G Biosciences (1 mentions)
Signalfire enhanced chemiluminescence ecl reagent, by Cell Signaling Technology (1 mentions)
Cytokeratin 8, by Abcam (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Ecl plus chemiluminescence reagent, by Cytiva (1 mentions)
Peripherin, by Santa Cruz Biotechnology (1 mentions)
Alpha enolase, by Santa Cruz Biotechnology (1 mentions)
Vdac1, by Santa Cruz Biotechnology (1 mentions)
Streptozocin stz, by Merck Group (1 mentions)
Cyt c, by Cell Signaling Technology (1 mentions)
Ip3r1, by Santa Cruz Biotechnology (1 mentions)
Cox 4, by Proteintech (1 mentions)
4 hne, by Abcam (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Brn3a, by Abcam (1 mentions)
6 protocols using grp75
1
Western Blot Analysis of Cellular Proteins
2
Comprehensive Protein Expression Analysis
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Western blot analysis was performed as described [30 (link)]. Fifteen μg total protein lysate was separated on 10% SDS-PAGE. Proteins were then transferred to a PVDF membrane (Amersham Biosciences, Piscataway, NJ). The membrane was blocked, incubated with primary antibodies overnight and secondary antibody for 1 h at room temperature. The membrane was developed with ECL Plus chemiluminescence reagent (Amersham Biosciences). The primary antibodies used in this study were against actin (Santa Cruz, CA), peripherin (Santa Cruz, CA), cytokeratin-8 (Abcam, MA), aldose reductase (Abcam, MA), alpha-enolase (Santa Cruz, CA), Grp75 (Cell Signaling Technology, MA), phosphoglycerate mutase (Santa Cruz, CA), F1 ATPase (Santa Cruz, CA), SCO2 (Santa Cruz, CA) and OGDH (Santa Cruz, CA).
3
Detailed Reagents and Materials Protocol
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Glucose, streptozocin (STZ), and Evans blue were purchased from Sigma-Aldrich (St. Louis, MO, USA), while advanced glycosylation end products (AGEs) were acquired from BioVision (Palo Alto, CA, USA). Tunicamycin (TUN) was obtained from Abcam (Cambridge, CA, USA), while 4-phenylbutyric acid (4-PBA), BAPTA-AM, and Protein A/G magnetic beads were purchased from MedChemExpress (Lowell, NJ, USA). Cell counting kit-8 (CCK8), JC-1 assay kit, Calcein/PI assay kit, MPTP assay kit, Mito-Tracker Green, ER-Tracker Red, and a mitochondria isolation kit were purchased from Beyotime (Nantong, China). On the other hand, the TUNEL assay kits were purchased from Roche (Basel, Switzerland). MitoSOX™ Red, DAPI, ECL kits, and goat anti-mouse/rabbit IgG (H+L, Alexa Fluor 555/488) were acquired from Thermo Fisher (Waltham, CA, USA). We used primary antibodies including GRP75, Cyt c, Bcl-xl, Bax, cleaved caspase-3, and CHOP (Cell Signaling Technology, Boston, MA, USA); 4-HNE and Brn3a (Abcam, Cambridge, CA, USA); IP3R1, VDAC1, VEGF, 8-OHDG, and 3-NT (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and COX IV, Bcl-2, and β-actin (Proteintech, Wuhan, China). More detailed information about the materials and reagents is offered in Table S1 .
4
Protein Isolation and Western Blot Analysis
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Total
protein was isolated from the cell
lysates following transfection (72 h). Protein samples were prepared
by lysing the cells in ice-cold radioimmunoprecipitation assay buffer
(G-Biosciences) supplemented with protease and phosphatase inhibitors
(Millipore Sigma), which were diluted 1:10 as per the manufacturer’s
recommendations. Cell debris was removed by centrifugation at 16 000g at 4 °C, and protein concentrations were determined
using a PierceTM BCA kit (Thermo Fisher Scientific). A sample (20–35
mg) of the supernatant protein was mixed with lithium dodecyl sulfate
buffer and dithiothreitol, incubated at 70 °C for 10 min and
resolved on a 4–12% Bis–Tris PAGE gradient gel before
being transferred to a poly(vinylidene difluoride) membrane. Following
transfer, the membrane was blocked in 5% skim milk for 1 h, washed
and incubated at 4 °C overnight with a rabbit primary mAb against
human GRP 78, GRP 94, GRP 75, or GAPDH (all purchased from Cell Signaling
Technology) at a 1:1000 dilution. The membrane was subsequently washed
and incubated with an anti-rabbit horseradish peroxidase-conjugated
secondary Ab (Cell Signaling Technology) for 1 h at room temperature
at 1:2000 dilution. The bands were visualized using a SignalFire ECL
reagent (Cell signaling Technology) on a ProteinSimple FluorChem E
imager.
protein was isolated from the cell
lysates following transfection (72 h). Protein samples were prepared
by lysing the cells in ice-cold radioimmunoprecipitation assay buffer
(G-Biosciences) supplemented with protease and phosphatase inhibitors
(Millipore Sigma), which were diluted 1:10 as per the manufacturer’s
recommendations. Cell debris was removed by centrifugation at 16 000g at 4 °C, and protein concentrations were determined
using a PierceTM BCA kit (Thermo Fisher Scientific). A sample (20–35
mg) of the supernatant protein was mixed with lithium dodecyl sulfate
buffer and dithiothreitol, incubated at 70 °C for 10 min and
resolved on a 4–12% Bis–Tris PAGE gradient gel before
being transferred to a poly(vinylidene difluoride) membrane. Following
transfer, the membrane was blocked in 5% skim milk for 1 h, washed
and incubated at 4 °C overnight with a rabbit primary mAb against
human GRP 78, GRP 94, GRP 75, or GAPDH (all purchased from Cell Signaling
Technology) at a 1:1000 dilution. The membrane was subsequently washed
and incubated with an anti-rabbit horseradish peroxidase-conjugated
secondary Ab (Cell Signaling Technology) for 1 h at room temperature
at 1:2000 dilution. The bands were visualized using a SignalFire ECL
reagent (Cell signaling Technology) on a ProteinSimple FluorChem E
imager.
5
Immunoblotting with Antibodies
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Primary and secondary antibodies for immunoblotting were used at dilutions of 1:2,000 and 1:10,000, respectively. OGT (AL-34), and OGA (345) were gracious gifts from the laboratory of Gerald Hart in the Department of Biochemistry and Molecular Biology at the University of Georgia. The other antibodies used were as follows: anti-O-linked N-acetylglucosamine antibody (RL2, Thermo Fisher, MA1-072), anti-GAPDH antibody (Abcam, ab9484), GRP75 (Cell Signaling Technology, D13H4), ATF5 (Abcam, ab184923), ATF4 (Cell Signaling Technology, D4B8), TOM20 (Santa Cruz Biotechnology, F-10, sc-17764), α-tubulin (Sigma-Aldrich, T5168), anti-chicken IgY-HRP (Sigma-Aldrich, A9046), goat anti-rabbit IgG-HRP (Bio-Rad, 170–6515), and goat anti-mouse IgG-HRP (Bio-Rad, 170–6516).
6
Kidney Tissue I/R Injury Protein Analysis
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Kidney tissues at 24 h after I/R were ground into homogenates in liquid nitrogen. Protein was extracted from tissues and cells using Radio Immunoprecipitation Assay (RIPA) buffer with protease inhibitors on ice. The concentration of protein was detected using Easy II Protein Quantitative Kit (TransGen Biotech). Proteins (50 μg) were separated on 12% polyacrylamide gels using electrophoresis. Then proteins were transferred to PVDF membranes. After blocking with nonfat milk, the membranes were incubated with primary antibodies (Mfn2, GRP78, PERK, p-PERK, GRP75, MICU1, IP3R1, MCU, VDAC1, and β-actin; Cell Signaling Technology, MA, USA) overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated IgG antibody at room temperature for 2 h. The bands were observed with ECL solution on chemiluminescence system (Bio-Rad). Protein relative expression was calculated by ImageJ software.
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