The protein concentration was quantified by BCA assay (ThermoFisher Scientific), and 20–40 µg of protein was separated in 8% PAGE gel. The proteins were transferred to a nitrocellulose membrane at 35 V for 3 h for CaV3.1 and CaV3.2 and 100 V for 1 h for pERK1/2. The protein transfer was confirmed by Ponceau staining, and the blots were washed and incubated with 5% non-fat milk (Sigma) for 1 h. The blots were incubated with primary antibodies: CaV3.1 (1:1000) (Alomone, Jerusalem, Israel) and CaV3.2 (1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4 °C. The following day, the blots were washed thrice with 1 × TBS + 0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1 × TBS-T and incubated with ECL substrate (Fisher scientific) for 5 min and exposed to X-ray film. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quantified using Image Studio Lite software.
Total erk1 2
Manufactured by Merck Group
Total ERK1/2 is a lab equipment product designed to measure the total levels of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in biological samples. ERK1/2 are important signaling proteins involved in cellular processes such as proliferation, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Stripping buffer, by Merck Group (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Total erk1 2, by Cell Signaling Technology (2 mentions)
Non fat milk, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Erbb2, by Cell Signaling Technology (1 mentions)
Erbb3, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Actin, by Merck Group (1 mentions)
Phospho erk1 2, by Merck Group (1 mentions)
4 protocols using total erk1 2
1
Western Blot Analysis of Ion Channels
2
Western Blot Analysis of Receptor Signaling
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Protein was extracted from cells using 1X RIPA buffer containing a Halt Protease and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Cell lysate proteins (30 μg) were separated on 4–12% NuPAGE Bis-Tris precast gel electrophoresis and transferred to iBlot 2 polyvinyl difluoride membranes (Invitrogen, Carlsbad, CA). The blots were incubated with the appropriate antibodies to detect the protein level of interest, and the immune complexes were visualized by GE Healthcare Amersham ECL WB detection system (ThermoFisher Scientific). Western blots were probed with antibodies against phosphor-ERBB2/HER-2 (Tyr1248) (Cell Signaling Technologies, Danvers, MA; CST# 2247, diluted 1:800), ERBB2 (CST# 2165, diluted 1:800), phosphor-ERBB3 (Tyr1197) (CST# 4561, diluted 1:800), ERBB3 (CST# 4754, diluted 1:800), phosphor-ERK1/2 (CST# 4370, diluted 1:1000), phosphor-p44/p42 MAPK (diluted 1:1000), total p44/42 MAPK, (diluted 1:1000), total ERK1/2 (CST# 9102, diluted 1:1000), phosphor-AKT S473 (CST# 9271, diluted 1:800), AKT (CST# 9272, diluted 1:800), phosphor-EGFR (CST# 3777, diluted 1:800), phosphor-p90RSK (CST# 11989, diluted 1:800), and Actin (Sigma, St. Louis, MO, diluted 1:10,000).
3
Protein Kinase Inhibitors and ELISA
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PD98059 (PD), SB203580 (SB) and SP6000125 (SP) were purchased from Calbiochem (San Diego, CA, USA). All enzyme-linked immunosorbent assay (ELISA) kits for phospho-p38 MAPK (Product No: CS0020), total p38 MAPK (Product No: PM0100), Phospho-JNK1&2 (Product No: CS0130), JNK 1&2 (Product No: CS01000), Phospho-ERK1/2 (Product No: CS 7177), total ERK1/2 (Product No: CS 7050), and P-selectin ELISA kit (Catalog Number RAB0426.) were from Sigma Aldrich.
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PD98059 (PD), SB203580 (SB) and SP6000125 (SP) were purchased from Calbiochem (San Diego, CA, USA). All enzyme-linked immunosorbent assay (ELISA) kits for phospho-p38 MAPK (Product No: CS0020), total p38 MAPK (Product No: PM0100), Phospho-JNK1&2 (Product No: CS0130), JNK 1&2 (Product No: CS01000), Phospho-ERK1/2 (Product No: CS 7177), total ERK1/2 (Product No: CS 7050), and P-selectin ELISA kit (Catalog Number RAB0426.) were from Sigma Aldrich.
4
Western Blot Analysis of Ion Channels
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The protein concentration was quanti ed by BCA assay (ThermoFisher Scienti c), and 20-40 µg of protein was separated in 8% PAGE gel. The proteins were transferred to a nitrocellulose membrane at 35 V for 3 h for Ca V 3.1 and Ca V 3.2 and 100 V for 1 h for pERK1/2. The protein transfer was con rmed by Ponceau staining, and the blots were washed and incubated with 5% non-fat milk (Sigma) for 1 h. The blots were incubated with primary antibodies: Ca V 3.1 (1:1000) (Alomone, Jerusalem, Israel) and Ca V 3.2
(1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4°C. The following day, the blots were washed thrice with 1x TBS +0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1x TBS-T and incubated with ECL substrate (Fisher scienti c) for 5 min and exposed to X-ray lm. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quanti ed using Image Studio Lite software.
(1:500) (Alomone), pERK1/2 (1:5000) (Cell signaling, London, UK), total ERK1/2 (1:5000) (cell signaling) and beta-actin (1:5000) (Abcam, Cambridge, UK) overnight at 4°C. The following day, the blots were washed thrice with 1x TBS +0.1% Tween 20 (TBS-T) for 10 min and then incubated with anti-rabbit secondary antibody conjugated with horse radish peroxidase for 1 h at room temperature. The blots were washed thrice with 1x TBS-T and incubated with ECL substrate (Fisher scienti c) for 5 min and exposed to X-ray lm. In order to probe for total ERK1/2 and beta-actin, the blots were stripped with stripping buffer (Sigma) for 30 min at RT under dark. Protein band density was quanti ed using Image Studio Lite software.
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