In order to identify the different interacting partners for sGRP78, control and cytokine-exposed INS-1E cells were subjected to a membrane fractionation isolation as described above. Subsequently, membrane proteins were eluted in 2 mM D-biotin (in PBS, ThermoFisher scientific) by centrifugation at 2000 rpm for 4 min at 4 °C. The eluate was then incubated with 6 µg of anti-GRP78 antibody (#sc-1050, Santa Cruz Biotechnology) overnight at 4 °C, followed by incubation with 30 µl A/G PLUS-agarose beads (#sc-2003, Santa Cruz Biotechnology) for 4 h at 4 °C. The beads were washed three times with pre-urea buffer (50 mM Tris; pH 8.5, 1 mM EGTA, and 75 mM KCl), followed by elution in urea buffer (7 M urea, 20 mM Tris; pH 7.5 and 100 mM NaCl). The proteins obtained were identified by LC-MS/MS on an Orbitrap Q Exactive (ThermoFisher scientific) as previously described3 (link).
Anti grp78 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-GRP78 antibody is a research tool used to detect and study the GRP78 protein, also known as the 78 kDa Glucose-Regulated Protein. GRP78 is a molecular chaperone that plays a crucial role in protein folding and the unfolded protein response within the endoplasmic reticulum. This antibody can be used in various research applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to facilitate the investigation of GRP78 and its cellular functions.
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Lab products found in correlation
D biotin, by Thermo Fisher Scientific (1 mentions)
A g plus agarose beads, by Santa Cruz Biotechnology (1 mentions)
Q exactive orbitrap, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose immunoprecipitation reagent, by Santa Cruz Biotechnology (1 mentions)
Anti perk antibody, by Santa Cruz Biotechnology (1 mentions)
Situ cell death detection kit, by Roche (1 mentions)
Anti nrf2 antibody, by Santa Cruz Biotechnology (1 mentions)
Edu staining kit, by RiboBio (1 mentions)
Anti rabbit fitc, by Abcam (1 mentions)
Envisiontm, by Agilent Technologies (1 mentions)
Dmi4000d microscope, by Leica camera (1 mentions)
Anti gfp antibody, by Merck Group (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Anti hsp90α antibody, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Protein g sepharose, by Roche (1 mentions)
Secondary antibody conjugated to horseradish peroxidase, by Boster Bio (1 mentions)
Anti p erk antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
8 protocols using anti grp78 antibody
1
Identifying sGRP78 Interacting Partners
2
GRP78 Immunoprecipitation and PERK Detection
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MSCs were lysed with a lysis buffer (1% Triton X-100 in 50 mM Tris-HCl [pH 7.4] containing 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 2.5 mM Na4PO7, 100 mM NaF, and protease inhibitors). Cell lysates (300 μg) were mixed with anti-GRP78 antibody (Santa Cruz Biotechnology). The samples were incubated for 4 h, mixed with Protein A/G PLUS-Agarose Immunoprecipitation Reagent (Santa Cruz Biotechnology), and then incubated for an additional 12 h. The beads were washed four times, and the bound protein was released from the beads by boiling in SDS-PAGE sample buffer for 5 min. The precipitated proteins were analyzed by western blotting with anti-PERK antibody (Santa Cruz Biotechnology).
3
Immunofluorescence and Apoptosis Analysis of H9c2 Cells
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Immunofluorescence was implemented in the 4% paraformaldehyde‐fixed H9c2 cells. And then cells were incubated with anti‐GRP78 antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, CA) and anti‐Nrf2 antibody (1:400; Santa Cruz Biotechnology) overnight at 4°C. After washed five times with PBS for 1 hr the next day, H9c2 cells were incubated with the secondary antibodies (AlexaFluor488 and AlexaFluor594, 1:200) for 2 hr at room temperature. Then the cells were washed with PBS again for five times for 1 hr before being stained by 4′,6‐diamidino‐2‐phenylindole for 2 min. After three further washes, the dishes were observed with a fluorescence microscope. Negative controls were prepared by omitting the primary antibodies. For EdU (5‐ethynyl‐2'‐deoxyuridine) assay, H9c2 cells were seeded in 96‐well plates and treated with EdU 6 hr. Subsequently EdU incorporated cells were subjected to staining with a commercial EdU staining kit (#C10310‐2; RiboBio, Guangzhou, China).
The apoptosis profile of the H9c2 cells was evaluated using a commercially available situ cell death detection kit (Roche Diagnostics) to test the terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL).
The apoptosis profile of the H9c2 cells was evaluated using a commercially available situ cell death detection kit (Roche Diagnostics) to test the terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL).
4
GRP78 Interactome Identification
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For immunoprecipitation, extracted membrane protein was incubated with home‐made magnetic particles conjugated with anti‐GRP78 antibody (Santa Cruz, CA, USA) at 4°C overnight to pull down GRP78 and proteins interacted with it. Magnetic particles were then washed two times with lysis buffer, and the bound proteins were eluted with Laemmli SDS buffer supplemented with 50 mM dithiothreitol, followed by incubation for 10 min. at 100°. Then, the precipitated proteins were analysed by Western blotting.
5
Immunohistochemistry of GRP78 in Human Lung
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Formalin-fixed human lung tissue sections were de-paraffinized in a xylene series and rehydrated through a decreasing ethanol series for immunohistochemistry. The slides were pre-treated by microwave in citrate buffer (100 mM, pH 7.0) for 10 minutes, washed 3x with 1x PBS and 0.1% tween (TBS). Slides were incubated overnight at 4 °C in an anti-Grp78 antibody dilution (1:100) from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). EnVisionTM (DAKO, Bollschweil, Germany) was used for staining and detection, according to the manufacturer’s instructions. A Leica DMI 4000 D microscope was used to acquire images. For double immunofluorescence, tissue sections were incubated with primary antibodies specific to GRP78 (1:100) or SP-C (1:100) (surfactant protein C) (both: Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies used were anti-goat Cy3 labelled to detect GRP78, and anti-rabbit FITC labelled to detect SP-C (Abcam, USA); both secondary antibodies were diluted 1:1000.
6
Immunoprecipitation and Western Blotting of BiP and ATF6
7
Immunoprecipitation of Hsp90α and GRP78
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Conditioned media from A549 and H1299 cells were collected and incubated with anti-Hsp90α antibody (Abcam, Cambridge, MA, USA, ab2928, 1:2,000 dilution), anti-GRP78 antibody (Santa Cruz, Dallas, TX, USA, SC-1050, 1:1,000 dilution), and protein G-Sepharose (Roche, Basel, Switzerland) overnight at 4°C with constant rotation. Before analyzing by immunoblot, we washed immuno-complexes three times with IP lysis buffer. Antibodies against PKM2 (#4053, 1:1,000 dilution) and β-actin (#3700, 1:1,000 dilution) were obtained from Cell Signaling Technologies (Danvers, MA).
8
Protein Extraction and Western Blot Analysis
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Proteins were extracted in 100 µl of SDS lysis buffer (10
mM EDTA, 25 mM Tris/HCl, pH 7.4, 95 mM NaCl, 2%
SDS) with protease inhibitors (Sigma). Protein content was measured by the BCA
method (Pierce) according to the manufacturer’s instructions. Total
proteins (40 µg) were separated by SDS/PAGE (10% gel).
Then the protein was blotted on to PVDF membrane (Bio–Rad) at 70 V for 65
min. Subsequently, the membranes were incubated with primary antibodies,
including anti-PERK antibody (Cell Signaling), anti-IRE1 antibody (R&D
Systems), anti-GRP78 antibody (Santa Cruz Biotechnology), anti-CHOP antibody
(R&D Systems), anti-cleaved caspase-12 antibody (R&D Systems),
anti-cleaved caspase-3 antibody (Cell Signaling), anti-Bcl-2 antibody
(R&D Systems), and anti-GAPDH antibody (Sigma–Aldrich) overnight
at 4 °C. Then the membranes were incubated with secondary antibody
conjugated to horseradish peroxidase (Boster, Wuhan, China) for 1 h at
room temperature. The chemiluminescent signal was detected by the ECL Detection
Reagents (Amersham).
mM EDTA, 25 mM Tris/HCl, pH 7.4, 95 mM NaCl, 2%
SDS) with protease inhibitors (Sigma). Protein content was measured by the BCA
method (Pierce) according to the manufacturer’s instructions. Total
proteins (40 µg) were separated by SDS/PAGE (10% gel).
Then the protein was blotted on to PVDF membrane (Bio–Rad) at 70 V for 65
min. Subsequently, the membranes were incubated with primary antibodies,
including anti-PERK antibody (Cell Signaling), anti-IRE1 antibody (R&D
Systems), anti-GRP78 antibody (Santa Cruz Biotechnology), anti-CHOP antibody
(R&D Systems), anti-cleaved caspase-12 antibody (R&D Systems),
anti-cleaved caspase-3 antibody (Cell Signaling), anti-Bcl-2 antibody
(R&D Systems), and anti-GAPDH antibody (Sigma–Aldrich) overnight
at 4 °C. Then the membranes were incubated with secondary antibody
conjugated to horseradish peroxidase (Boster, Wuhan, China) for 1 h at
room temperature. The chemiluminescent signal was detected by the ECL Detection
Reagents (Amersham).
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