The total protein of hypothalamus from each mouse was extracted using RIPA Lysis buffer (Biomiga, Santiago, CA, USA) and the BCA protein assay kit (Termo Fisher Scientific) was used to determine the protein concentration. The protein expressions of apelin and APJ were measured by western blot and the procedure was performed as previously described [56 (link)]. The 12% SDS-PAGE was selected to conduct transmembrane according to relative molecular weight of apelin and APJ, and then proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. PBST with 5% nonfat milk was used to block the membranes, and the primary antibodies were apelin (M-77) (SC-33805, 1:100 dilution, Santa Cruz, Dallas, TX, USA), Apelin receptor Antibody (APJ) (PA5-21306, 1:500 dilution, Thermo Scientific) and β-Tubulin Monoclonal Antibody (A01030, 1:5000 dilution, Abbkine, Inc., Redlands, CA, USA). The SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific) was used to develop the membranes and the Tanon-5200 analysis system (Tanon, Shanghai, China) was used for exposure, then the optical density of protein bands were read by Tanon Gis software.
Ripa lysis buffer
Manufactured by Biomiga
Sourced in United States
RIPA Lysis buffer is a commonly used cell lysis solution designed for the extraction of proteins from cells or tissues. It is a detergent-based buffer that helps to solubilize and denature proteins, allowing for their subsequent analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Ab121013, by Abcam (1 mentions)
Ab106134, by Abcam (1 mentions)
Ab125066, by Abcam (1 mentions)
Ab214362, by Abcam (1 mentions)
Ab76582, by Abcam (1 mentions)
Ab178847, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
Ab184699, by Abcam (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P stat3 705, by Cell Signaling Technology (1 mentions)
P stat3 727, by Cell Signaling Technology (1 mentions)
Las4010, by GE Healthcare (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
5 protocols using ripa lysis buffer
1
Analyzing Apelin and APJ Protein Expression
2
Hippocampal GFAP and NR2B Protein Expression
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Protein levels of the GFAP and NR2B in the hippocampus were identified by WB. Hippocampal tissues were used to prepare the total proteins with RIPA Lysis buffer (Biomiga, Santiago, CA, USA). According to the molecular weight of the target protein GFAP (50 kDa) and NR2B (166 kDa), 12% and 5% SDS-PAGE gels were selected, and then proteins were transferred onto polypropylene fluoride (PVDF) membranes. The 5% nonfat milk was used to block the membranes and dilute antibody, and the primary antibodies were anti-GFAP antibody (60190-1-lg, Mouse polyclonal to GFAP, diluted 1:5,000; Proteintech Group, Rosemont, IL, USA), anti-GRIN2B antibody (21920-1-AP, rabbit polyclonal to NR2B, diluted 1:500; Proteintech Group, Rosemont, IL, USA) and β-actin monoclonal antibody (66009-1lg, mouse monoclonal to β-actin, diluted 1:5,000; Proteintech Group, Rosemont, IL, USA). The enhanced chemiluminescence (ECL) detection reagent and Thermo Fisher Scientific was used to develop the membranes for 2 min, and then the Tanon-5200 system (Tanon, Shanghai, China) was used for exposure. The intensity of the protein band was read by Tanon Gis software (Tanon).
3
Western Blot Analysis of TPH2 and IDO1
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The expression of the TPH2 and IDO1 proteins in the hippocampus was detected by Western blot. The hippocampal tissues were used to prepare the total proteins with RIPA Lysis buffer (Biomiga, Santiago, CA, USA). The 10% SDS-PAGE gels were selected according to relative molecular weight of TPH2 (56 kDa) and IDO1 (45 kDa), and then proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The 5% nonfat milk was used to block the membranes, and the primary antibodies were anti-TPH2 antibody (ab121013, goat polyclonal to TPH2, diluted 1:500; Abcam, San Francisco, CA, USA), anti-indoleamine 2,3-dioxygenase antibody (ab106134, rabbit polyclonal to indoleamine 2,3-dioxygenase, diluted 1:50; Abcam) and β-actin monoclonal antibody (MA1-140, mouse monoclonal to β-actin, diluted 1:3,000; Thermo Fisher Scientific). The enhanced chemiluminescence (ECL) detection reagent (Thermo Fisher Scientific) was used to develop the membranes for 1 minute, and then the Tanon-5200 system (Tanon, Shanghai, China) was used for exposure. The optical density of protein bands was read by Tanon Gis software (Tanon).
4
Hippocampal Protein Analysis by Simon WB
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The total protein of hippocampal tissues was extracted using the RIPA Lysis Buffer (Biomiga, Santiago, USA). Then the protein levels of GPX4 (Anti-Glutathione Peroxidase 4 antibody [EPNCIR144], ab125066, 1:500 dilution, Abcam, San Francisco, USA), PEBP1 (Anti-RKIP antibody [EPR2875Y], ab76582, 1:500 dilution, Abcam, San Francisco, USA), IBA1 (Anti-Iba1 antibody [EPR16589], ab178847, 1:100 dilution, Abcam, San Francisco, USA), GFAP (Anti-GFAP antibody, ab7260, 1:1000 dilution, Abcam, San Francisco, USA), total ERK1/2 (t-ERK1/2) (Anti-ERK1 + ERK2 antibody [EPR17526], ab184699, 1:1000 dilution, Abcam, San Francisco, USA) and phosphorylated ERK1/2 (p-ERK1/2) (Anti-ERK1 + ERK2 [phospho T202 + Y204] antibody, ab214362, 1:100 dilution, Abcam, San Francisco, USA) were measured by Simon Western Blot Analysis (ProteinSimple, California, USA), a capillary-based automated system with a standard protocol.51 (link)
5
Western Blot for Protein Expression
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Cells or tumor tissues were collected and suspended in RIPA lysis buffer (Biomiga, Inc.) containing a cocktail of proteinase inhibitors (Roche). The protein concentration was quantified using the bicinchoninic acid (BCA) assay kit (Thermo scientific, Inc.) to ensure that equal amounts of protein from different subpopulations were loaded into the gel. The proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane. Primary antibodies against murine GZMM (P-15, Santa Cruz Biotechnology, Inc.); E-cadherin, N-cadherin, vimentin, AKT, p-AKT, ERK1/2, p-ERK1/2, STAT3, p-STAT3705, p-STAT3727 (Cell Signaling Technology), a-tubulin (ZSGB-BIO), and β-actin (ZSGB-BIO) were used at 1:1000 dilution and incubated with the membranes at 4°C overnight. The reaction was revealed by horseradish peroxidase (HRP)-coupled secondary reagents (Pierce, Rockford, IL, USA), developed by enhanced chemiluminescence (ECL) (Applygen Technologies Inc.) according to the manufacturer's instructions, and subjected to exposure using LAS4010 (General Electric Company).
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