Anti grp78

Manufactured by Affinity Biosciences

Sourced in China

Anti-GRP78 is a laboratory reagent used for the detection and quantification of GRP78 (glucose-regulated protein 78), a chaperone protein involved in the unfolded protein response. It can be used in various techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the expression and localization of GRP78 in biological samples.

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GRP78 (4 citations)

4 protocols using anti grp78

Western Blot Analysis of ER Stress Proteins

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Following the aforementioned treatments, H9c2 cells were washed thoroughly with ice-cold PBS solution, and RIPA buffer (Beyotime Institute of Biotechnology) was then added to the wells and incubated for 30 min on ice. Protein was quantified using a BCA assay. A total of 30 µg proteins/lane were separated by 8% SDS-PAGE and transferred to PVDF membranes at 4˚C and 200 mA for 2 h. The membranes were blocked by 5% skimmed milk powder in TBS with Tween-20 (TBS-T) solution for 2 h at room temperature and then incubated at 4˚C overnight with the following primary antibodies: Anti-CHOP (1:1,000; cat. no. DF6025; Affinity Biosciences), anti-GRP78 (1:1,000; cat. no. AF5366; Affinity Biosciences), anti-caspase-12 (1:1,000; cat. no. AF5199; Affinity Biosciences) and mouse anti-β-actin (1:1,000; cat. no. T0022; Affinity Biosciences). A horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. 111-095-003; Jackson ImmunoResearch Laboratories, Inc.) was then added for 2 h at room temperature after the membranes were washed five times with TBS-T buffer. Finally, the membranes were washed with TBST, and signals were visualized with an enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology). The protein band densities were quantified with ImageQuant TL software (version 7.0; Cytiva).

Zhu Z., Ling X., Zhou H, & Zhang C. (2020). Dexmedetomidine at a dose of 1 µM attenuates H9c2 cardiomyocyte injury under 3 h of hypoxia exposure and 3 h of reoxygenation through the inhibition of endoplasmic reticulum stress. Experimental and Therapeutic Medicine, 21(2), 132.

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Western Blot Analysis of Stress Response Proteins in H9c2 Cells

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Following the treatments described in previous procedures, H9c2 cells were washed well with an ice-cold PBS solution, and then, we added a RIPA solution (Beyotime, China) to the wells and incubated the cells for 30 min on ice. Subsequently, the supernatants were collected after centrifugation of the lysates. The BCA method was used to measure the protein concentration. The proteins were separated by SDS-PAGE and transferred to PVDF membranes at 4 °C and 200 mA for 2 h. Then, the membranes were blocked in a TBST solution for 2 h at room temperature and incubated at 4 °C overnight with the following primary antibodies: anti-dual phospho-p38 MAPK (Thr¹⁸⁰ and Tyr¹⁸²) (Sigma, 1:1000), anti-p38 MAPK (Sigma, 1:1000), anti-CHOP (Affinity, 1:1000), anti-GRP78 (Affinity. 1:1000), anti-caspase12 (Affinity, 1:1000) and mouse anti-β-actin (Affinity, 1:1000). A secondary antibody conjugated to horseradish peroxidase (Jackson, 1:2000) was added and incubated for 2 h at room temperature. Finally, the membranes were washed in TBST, and the signals were visualized with an enhanced chemiluminescence detection kit (ECL, Beyotime, China). The density of the protein bands was quantified by IQuantTL (GE Healthcare, USA). Relative protein expression was calculated with normalization to β-actin expression.

Zhu Z., Ling X., Zhou H., Zhang C, & Yan W. (2020). Dexmedetomidine Attenuates Cellular Injury and Apoptosis in H9c2 Cardiomyocytes by Regulating p-38MAPK and Endoplasmic Reticulum Stress. Drug Design, Development and Therapy, 14, 4231-4243.

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Protein Expression Analysis by Western Blot

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Western blotting analysis was performed as described previously [24 (link)]. The antibodies used in this study were all purchased from Affinity Biosciences, as follows: anti-GRP78 (Cat# AF5366), anti-DDIT/CHOP (Cat# DF6025), anti-mTOR (Cat# AF6308), anti-phospho(P)-mTOR (Cat# AF3308), anti-P-p70S6 (Cat# AF6226), anti-P-p70S6 kinase (Thr389/Thr412) (Cat# AF3228), anti-pan-Akt1/2/3 (Cat# AF6261), anti-P-pan-Akt1/2/3 (Ser473) (Cat# AF0016), anti-P-IRE1 (Ser724) (Cat# AF7150), anti-JNK1/2/3 (Cat# AF6318), anti-P-JNK1/2/3 (Thr183+Tyr185) (Cat# AF3318), anti-caspase-3 (Cat# AF6311), and anti-BCL-2 (Cat# AF6139) antibodies. Anti-beta actin (β-actin) (Cat# AF7018) and anti-COX IV (Cat# AF5468) antibodies were used as loading controls.

Bai G., Wang H, & Cui N. (2022). mTOR pathway mediates endoplasmic reticulum stress-induced CD4+ T cell apoptosis in septic mice. Apoptosis, 27(9-10), 740-750.

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Western Blot Analysis of mTOR Pathway

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CD4⁺ T cells lysate was prepared by lysing in RIPA buffer containing protease inhibitors. Protein concentrations were estimated (BCA protein assay kit). After that, the protein samples were loaded onto each lane by SDS-PAGE for electrophoresis and transferred to 0.45 μM PVDF membrane (Millipore, MA, USA). Next, PVDF membranes were blocked with 5% skimmed milk in TBS-Tween for 60 min, followed by incubation with antibody at 4°C for 12-16 h. The following primary antibodies are as follows: anti-mTOR (Cat#AF6308, dilution rate 1 : 1000), anti-P-mTOR (Cat#AF3308, 1 : 1000), anti-P-p70s6k (Cat#AF3228, 1 : 1000), anti-p70s6k (Cat#AF6226, 1 : 1000), anti-4EBP (Cat#AF6432, 1 : 1000), anti-caspase-3 (Cat#AF6311, 1 : 1000), anti-Bcl-2 (Cat#AF6139, 1 : 1000), anti-Bax (Cat#AF0120, 1 : 1000), anti-GRP78 (Cat#AF5366, 1 : 1000), anti-CIRP (Cat#DF2643, 1 : 1000), anti-CHOP (Cat#DF6025, 1 : 1000), anti-CIRP (Cat#AF5366, 1 : 1000), and anti-actin-β (Cat#AF7018, 1 : 3000) were purchased from Affinity Biosciences (Jiangsu, China). Anti-P-4EBP (anti-eIF4EBP1, ab27792) was from Abcam (CA, USA). After washing with TBS-Tween 3 times, the membranes were incubated with a goat anti-rabbit IgG antibody (1 : 5000, Affinity Biosciences) at 25°C for 1 h, and the chemiluminescence signals were detected using an electrochemiluminescence detection system. Band densities were quantified by the ImageJ software.

Wang H., Chen J., Bai G., Han W., Guo R, & Cui N. (2022). mTOR Modulates the Endoplasmic Reticulum Stress-Induced CD4+ T Cell Apoptosis Mediated by ROS in Septic Immunosuppression. Mediators of Inflammation, 2022, 6077570.

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