Western blotting was performed as described in a prior study.17 (link) Hypothalami and testicles were washed twice in ice-cold phosphate-buffered saline (PBS), and then dissociated in radio-immunoprecipitation assay (RIPA) buffer. The samples were then centrifuged. 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 50 μg of total protein from each sample, which was then transferred to membranes. After blocking, membranes were incubated with anti-Leptin-R antibody (AF497, diluted 1:1000; R&D Systems, Minneapolis, MN, USA), anti-kisspeptin antibody (ab19028, diluted 1:100; Abcam, Cambridge, UK), and anti-GPR54 antibody (abs136643, diluted 1:100; Absin, Shanghai, China). Membranes were then washed and incubated with secondary antibodies (diluted 1:5000; ZSGB-BIO, Beijing, China). Detection was performed by chemiluminescence using ECL solution (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was tested at least in triplicate.
Ab19028
Manufactured by Abcam
Sourced in United States
Ab19028 is a mouse monoclonal antibody targeting Protein X. It is intended for use in immunoassay and other antibody-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Ez ecl enhanced chemiluminescence detection kit, by Sartorius (2 mentions)
Af497, by R&D Systems (1 mentions)
Ecl solution, by Thermo Fisher Scientific (1 mentions)
Ab46020, by Abcam (1 mentions)
Ab100896, by Abcam (1 mentions)
Bs 0061r, by Bioss Antibodies (1 mentions)
Bsm 0978m, by Bioss Antibodies (1 mentions)
Ab32072, by Abcam (1 mentions)
Ab188224, by Abcam (1 mentions)
Ab97200, by Abcam (1 mentions)
5 protocols using ab19028
1
Western Blotting Analysis of Leptin-R, Kisspeptin, and GPR54
2
Western Blot Analysis of Cellular Proteins
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The cellular protein lysates were obtained from the GT1-7 cells. The expression levels of β-actin, GAPDH and tubulin were used as the internal controls. In brief, protein (40μg each) from cell lysates were subjected to gel electrophoresis on SDS-polyacrylamide gel, and separated proteins were transferred onto nitrocellulose membranes and probed with rabbit antiserum against c-Myc (ab32072, abcam), Lin28a (ab46020, abcam), Kiss1 (ab19028, abcam), p53 (#32,532, cell signaling technology), p21 (ab188224, abcam), GRP54 (ab100896, abcam), β-actin (bs-0061R, Bioss, Beijing, China), and GAPDH (bsm-0978M, Bioss, Beijing, China). Then, the goat anti-rabbit secondary antibody (BV-S8008, Biovol Biotech, Shanghai, China) was used to incubate the membranes and an enhanced chemiluminescence Western blotting substrate kit (Pierce Rockford, IL, USA) was used to detect the signals.
3
Quantitative Analysis of Kiss1 and GnRH1 Proteins
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Kiss1 and GnRH1 proteins were quantitatively analyzed by WB. Total protein was obtained using RIPA lysis buffer (Medium 20115ES60, YEASEN, Shanghai, China). BCA protein detection kit (20201ES76, YEASEN, Shanghai, China) was used to test protein concentration. Afterward, 40-60ug of total protein were separated by 15% SDS-PAGE, and it then was transferred to polyvinylidene uoride (PVDF) membranes. The membranes were locked with 5% (wt./vol) skimmed milk for 2 hours and probed with primary antibody overnight at 4 ℃. Next, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:4,000; CST, New York) for 2 hours. Subsequently, the greyscale images were obtained and quanti ed using the Image J software (NIH). The primary antibodies used in the assay contained rabbit polyclonal anti-Kisspeptin antibody (ab19028; 1:200 dilution; Abcam, Los Angeles, CA), anti-GnRH1 antibody (abs138073, 1:1,000 dilution, Absin, Shanghai, China), mouse monoclonal anti-GAPDH (T0004, A nity, Jiangsu, China) or rat polyclonal anti-Tubulin antibody (30302ES20, YEASEN, Shanghai, China).
4
Western Blot Analysis of Ovarian KISS1 and GAPDH
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The detection of ovarian KISS1 and the internal control GAPDH in all experiments was performed by western blot, following separation by SDS-PAGE on 15% polyacrylamide gels under reducing conditions. The proteins were transferred to nitrocellulose membranes, blocked with 5% milk for 1 h and probed with rabbit polyclonal anti-KISS1 antibody (ab19028, Abcam) in a 1:50 dilution overnight, a 1:40,000 dilution of rabbit polyclonal anti-GAPDH (antibody G9545, Sigma-Aldrich) or in a 1:10,000 dilution of rabbit polyclonal anti-FSH receptor (antibody NBP1-4630, Novus Biologicals, Littleton, CO, USA) for 1 h. The antibody complexes were detected using goat anti-rabbit IgG Fc (HRP) (ab97200, Abcam) at 1:10,000, and for chemiluminescence, an EZ-ECL Enhanced Chemiluminescence Detection Kit (Biological Industries, KBH, Israel) was used. Chemiluminescence was captured using a G-Box Syngene system (Syngene Headquarters, MD, USA).
5
Western Blot Analysis of Ovarian Proteins
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Detection of ovarian KP and the internal control GAPDH by western blot at different ages was performed, following separation by SDS-PAGE on 15% polyacrylamide gels under reducing conditions. The proteins were transferred to PVDF membranes, blocked with 5% milk for 1 h and probed with either rabbit polyclonal anti-KP antibody (ab19028, Abcam, Inc., Cambridge, MA, USA) in a 1:50 dilution overnight or in a 1:40 000 dilution of rabbit polyclonal anti-GAPDH (antibody G9545, Sigma-Aldrich Co.) for 1 h. The antibody complexes were detected using goat anti-rabbit IgG Fc (HRP) (ab97200, Abcam, Inc.) at 1:10 000, and for chemiluminescence, an EZ-ECL Enhanced Chemiluminescence Detection Kit (Biological Industries, KBH, Israel) was used. Chemiluminescence was captured using a G-Box Syngene system (Syngene Headquarters, MD, USA).
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