Protein samples of hypothalamus tissues from the pre-laying, early-laying, peak-laying, and ceased groups were extracted and determined using kits according to the manufacturer’s instructions (Applygen Co., LTD. Beijing, China). Equivalent amounts of total protein were subjected to 12 % SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking for 1 h at 37 °C, the membranes were incubated separately with rabbit Anti-Adiponectin antibody (bs-0471R, Beijing Biosynthesis Biotechnology Co., LTD), rabbit Anti-Adiponectin Receptor 1 antibody (bs-0610R, Beijing Biosynthesis Biotechnology Co., LTD), and rabbit anti-Adiponectin receptor 2 antibody (bs-0611R, Beijing Biosynthesis Biotechnology Co., LTD) overnight at 4 °C. The membranes were subsequently incubated with HRP-conjugated goat anti-rabbit antibody (bs-0295G-HRP, Beijing Biosynthesis Biotechnology Co., LTD) for 1 h at 37 °C. Finally, the bands were captured using a MicroChemi4.2 imaging system (DNR Bio-imaging Systems, Jerusalem, Israel), and densitometry analysis of protein bands was performed using GelQuant software (DNR Bio-imaging Systems, Jerusalem, Israel). β-actin (sc-47778, Santa Cruz Biotechnology, USA) was used as a reference protein to ensure equal loading. Triplicate experiments were performed for each sample.
Bs 0295g hrp
Manufactured by Bioss Antibodies
Sourced in China, United States
Bs-0295G-HRP is a horseradish peroxidase (HRP) conjugated secondary antibody. It is designed for use in immunoassays and other applications that require a labeled secondary antibody.
Automatically generated - may contain errors
Lab products found in correlation
Bs 0296g hrp, by Bioss Antibodies (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Bs 0061r, by Bioss Antibodies (4 mentions)
Bs 2188r, by Bioss Antibodies (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Ab181602, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Bioss Antibodies (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Gapdh, by Bioss Antibodies (2 mentions)
Ab125212, by Abcam (2 mentions)
Ab217344, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Microchemi 4.2 imaging system, by DNR Bio-Imaging Systems (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Gelquant software, by DNR Bio-Imaging Systems (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
35 protocols using bs 0295g hrp
1
Adiponectin Pathway Protein Expression
2
Quantifying Apoptosis Signaling Proteins in Hippocampal Tissue
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Frozen hippocampal samples were homogenated with triple detergent lysis buffer, containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 0.5% sodium deoxycholate, 100 mg/ml phenylmethylsulfonyl fluoride and 1 mg/ml aprotinin. Protein concentration was determined by the BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein samples were separated by 12% SDS-polyacrylamide gel electrophoresis, and then electronically transferred onto a nitrocellulose membrane. The membrane was blocked with 5% (w/v) fat-free milk in Tris-buffered saline containing 0.05% Tween-20, followed by incubation with rabbit anti-Bcl-2 (1:5,000; ab32314; Abcam, Cambridge, MA, UK), anti-caspase-3 (1:5,000; ab13847; Abcam), or anti-β-actin (ab8227; Abcam) polyclonal antibodies. at 4°C overnight. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:3,000; Cat. No., bs0295g-HRP; Beijing Biosynthesis Biotechnology Co., Ltd.). Protein bands were visualized with an enhanced chemiluminescence system (GelDocXR+; V140130; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein bands on the images and the relative protein expression were analyzed using Quantity One software (Version 4.62).
3
Western Blot Protein Extraction and Quantification
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Total proteins were extracted using ice-cold RIPA lysis buffer containing a protease inhibitor cocktail (Invitrogen) [17 (link)]. The BCA Protein Assay Kit (23225, Thermo Fisher Scientific, USA) was used to quantify proteins. Then, equal amounts of protein lysate (75 μg) were separated by 10% SDS-PAGE electrophoresis and transferred to a 0.45 μm polyvinylidene fluoride membrane (Millipore). Membranes were blocked using 5% skimmed milk in TBST (pH 7.6) at room temperature for 1 h, and incubated overnight at 4°C in the presence of the following primary antibodies: anti-Smad7 (1:1000, Ab190987, Abcam), and anti-GAPDH (1:10000, ab181602, Abcam). Subsequently, they were incubated with Goat Anti-rabbit IgG H&L/HRP secondary antibody (1:5000, bs-0295G-HRP, Beijing Biosynthesis Biotechnology Co. Ltd, Beijing, China). Enhanced chemiluminescence western blotting reagents (BL520A, Biosharp, Anhui, China) were used for protein detection. Protein density data were normalized to GAPDH levels (n = 6/group) and reported as fold change values ± standard error of the mean.
4
Western Blot Analysis of Apoptosis Regulators
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Western blot was used to measure the expression of proteins. Simply 50 μg total protein was extracted by lysis buffer and quantified with a bicinchoninic acid assay kit (Kaiji Biotech, Nanjing, China). The samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membrane. The membrane was then blocked with 5% skim milk in Tris-buffered saline with Tween solution. Subsequently, the membrane was sequentially incubated with primary antibodies (Rabbit anti-human Bcl-2, Bax, PPP2R2A, 1:2000; Abcam, Cambridge, United Kingdom) and secondary antibody (bs-0295G-HRP, 1:5000; Beijing Biosynthesis Biotechnology, Beijing, China). Finally, the bands were visualized by Enhanced Chemiluminescence Plus, and the integrated optical density was measured by software Lab Works version 4.5.
5
Protein Expression Analysis by Western Blot
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Took out the preserved sample, added RIPA protein lysate and protease inhibitor PMSF, fully lysed, 12,000 × g, centrifuge at 4 °C for 15min. Next, collected the supernatant, extracted the total protein, and used BCA protein detection kit (BioTek) to detect the protein concentration. Then added the collected protein supernatant to 5 × Loading Buffer, fully mixed, denature at 98 °C for 15min and store at -20 °C. The Western blot technique (Xiao et al., 2018 (link)) developed by our laboratory was used to separate the protein by polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Atlanta, GA, USA). The electrotransfer solution was washed away with 1× trimethylaminomethane buffer saline (TBST) and was sealed with TBST which contained 5% skimmed milk powder for 1 h. The antibody was incubated with rabbit polyclonal antibody StAR (bs-3570R, 1:500, Bioss) P450scc (bs-10099R, 1:500, Bioss) PGR (bs-23376R, 1:500, Bioss) with β-actin (bs-0061R, 1:3,000, Bioss) as an internal reference, overnight at 4 °C, after washing, goat anti-rabbit secondary antibody (bs-0295G-HRP, Bioss) at a dilution ratio of 1:3,000, at 37 °C for 1 h. Immune complexes were detected with enhanced chemiluminescence solution (Abnova, Taibei, Taiwan), and the expression was quantified using ImageJ (National Institutes of Health).
6
Immunohistochemical Analysis of Metabolic and Immune Markers
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Primary human antibodies for lactate dehydrogenase A (LDHA) (DF6280), phosphoglycerate kinase 1 (PGK1) (DF6722), enolase α (ENO1) (DF6191), β-catenin (AF6266) and c-Myc (AF0358) were purchased from Affinity Biosciences Ltd., Jiangsu, China. Primary human antibodies of S100A9 (bs-2697R), cluster of differentiation 3 receptor (CD3) (bs-0765R), cluster of differentiation 4 receptor (CD4) (bs-0647R), cluster of differentiation 8 receptor (CD8) (bs-4790R) and Forkhead/winged-helix transcription factor P3 (FOXP3) (bs-0269R) were obtained from BIOSS biosciences, Ltd., Beijing, China. Horseradish peroxidase (HRP)-conjugated secondary antibodies were also purchased from BIOSS (bs-0295G-HRP).
7
Western Blot Protein Analysis Protocol
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Cells were washed with pre‐ice PBS and lysed with RIPA buffer for 30 minutes followed by centrifugation to collect the supernatant. The concentration of proteins was measured using the BCA protein assay kit. Proteins were separated by SDS‐PAGE at 100 V for 2 hours and wet transferred to the PVDF membranes at 350 mA for 1.5 hours. Then, the membrane was blocked with 5% skimmed milk at room temperature for 1 hour followed by incubation with primary antibodies (anti‐FOXO3a antibody, 1:2500; anti‐PUMA antibody, 1 µg/mL, and anti‐β‐actin antibody, 1:200） overnight at 4°C. After that, the membrane was washed with TBST three times and incubated with HRP‐conjugated second antibody (bs‐0295G‐HRP, 1:3000; Bioss, China) at room temperature for 2 hours. At last, the ECL reagent was added to visualize the proteins and band densities were determined by the ImageQuant TL software (GE Healthcare, USA).24 Every experiment was performed in triplicate.
8
Western Blot Analysis of NEDD4L, ENaC-α, and GAPDH
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Radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) was used for the extraction of proteins, and the bicinchoninic acid (BCA) method (Beyotime Institute of Biotechnology) was used for the measurement of concentrations. Proteins (40 µg/lane) were loaded on 8% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked for nonspecific binding for 2 hours by incubation with 5% fat-free dry milk in 100 mM of Tris-buffered saline plus 0.1% Tween-18 (TBST). Primary antibodies included rabbit anti-NEDD4L antibody (ab46521; 1:1,000; Abcam), rabbit anti-ENaC-α antibody (ab214192; 1:1,000; Abcam), and rabbit anti-GAPDH antibody (KGAA002; 1:1,000; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The membrane was incubated with primary antibodies overnight at 4 ℃ and then washed with TBST 3 times. The membrane was then incubated with HRP-conjugated goat-anti-rabbit secondary antibody (bs-0295G-HRP; 1:2,000; Bioss, Woburn, MA, USA) for 1.5 hours at room temperature. The enhanced chemiluminescence detection system (ChemiDoc XRS + Imaging System; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to visualize the bands of antibody-detected proteins.
9
ZIK1 Protein Expression Analysis
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Cells were inoculated in a 6-well plate, transfected with plasmid and placed in a cell culture incubator with a constant temperature and humidity for 48 h, and cells were then collected. Cells were lysed with radioimunoprecipitation assay buffer (Beyotime Institute of Biotechnology) and a BCA kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration, followed by denaturation, electrophoresis separation, membrane transfer, washing and sealing (11 (link)). Next, a ZIK1 primary antibody (dilution, 1:1,000; cat. no. bs-19266R; Shanghai Bioplus Biotech Co., Ltd.) was added for overnight incubation at 4°C, followed by the addition of a secondary antibody (dilution, 1:1,000; cat. no. bs-0295G-HRP; BIOSS Antibodies Co., Ltd.). The blot was finally developed using enhanced chemiluminescence reagents (Amersham; Cytiva) and GAPDH (dilution, 1:10,000; cat. no. ab181602; Abcam) was used as the internal reference.
10
Hippocampal Protein Expression Analysis
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Rat-isolated hippocampal regions were analyzed using Western blotting. Briefly, RIPA lysis buffer (Solarbio, Beijing, China) was used to homogenize the tissues. Total protein (50 μg) was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). Following incubation in blocking buffer (TBS with 5% nonfat milk and 0.1% Tween 20), the membranes were probed with primary antibodies directed against SHANK3 (1:1500, ab93607, Abcam, Cambridge, UK), GKAP (1:1000, ab93611, Abcam, Cambridge, UK), PSD95 (1:1000, ab18258, Abcam, Cambridge, UK), and GAPDH (1:2000, BS-2188 R, Bioss, Beijing, China). Goat anti-mouse IgG-HRP (1:3000, BS-0296G-HRP, Bioss) and goat anti-rabbit IgG-HRP (1:3000, BS-0295G-HRP, Bioss) were used as the secondary antibodies.
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