Testicular tissues at different development stages were homogenized and lysed using a radio immunoprecipitation assay (RIPA) protein extraction kit (Solarbio, Beijing, China), according to the operating instructions. Protein concentrations within the testis samples were determined using a commercial bicinchoninic acid (BCA) Protein Assay kit (Beyotime, Shanghai, China). Twenty micrograms of the denatured protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) blotting membranes (Beyotime, Shanghai, China). After blocking in phosphate buffered saline tween-20 (PBST) containing 5% non-fat milk, the membranes were incubated overnight at 4 °C with either rabbit anti-BOLL polyclonal antibody (1:500, Bioss, Beijing, China) or anti-beta-actin polyclonal antibody (1:1500, Bioss, Beijing, China). After washing, the membranes were incubated with goat anti-rabbit IgG/HRP antibody (1:5000, Bioss, Beijing, China). Enhanced chemiluminescence signals were visualized in an X-ray room. This experiment was biologically repeated three times. Band intensities were quantified using AlphaEaseFC software (Protein Simple, Santa Clara, CA, USA). The BOLL protein expression results, from an average across tissues, were presented as bar charts.
Goat anti rabbit igg hrp antibody
Manufactured by Bioss Antibodies
Sourced in China
The Goat anti-rabbit IgG/HRP antibody is a secondary antibody that binds to rabbit primary antibodies. The antibody is conjugated with horseradish peroxidase (HRP), which can be used for colorimetric or chemiluminescent detection in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa protein extraction kit, by Solarbio (4 mentions)
Polyvinylidene difluoride pvdf blotting membranes, by Beyotime (3 mentions)
Anti beta actin polyclonal antibody, by Bioss Antibodies (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab181602, by Abcam (1 mentions)
Ecl chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Ecl western blotting substrate, by Solarbio (1 mentions)
P akt, by Wanlei (1 mentions)
Grp78, by Wanlei (1 mentions)
Atf 4, by Wanlei (1 mentions)
β actin, by Bioss Antibodies (1 mentions)
P ire1, by Bioss Antibodies (1 mentions)
Cleaved caspase 3, by Wanlei (1 mentions)
Bcl 2, by Wanlei (1 mentions)
P erk, by Wanlei (1 mentions)
Caspase 3, by Wanlei (1 mentions)
8 protocols using goat anti rabbit igg hrp antibody
1
BOLL Protein Expression in Developing Testis
2
Protein Expression Analysis in ADMSCs and Knee Tissue
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Total protein was extracted from ADMSCs and knee tissue samples in different groups. Protein concentration was determined using a BCA protein assay kit (23227; Thermo Fisher Scientific, Inc.). Protein samples were subjected to SDS-PAGE and then transferred to a PVDF membrane. After addition of type II collagen (ab34712), aggrecan (ab3778), notch1 (ab52627), Jagged1 (ab7771), and GAPDH (ab181602; all Abcam) protein samples were incubated at 4°C overnight and washed with PBS. After addition of secondary antibody (goat anti-rabbit IgG/HRP antibody; 1:2,000; Bioss, Beijing, China), protein samples were incubated at 37°C for 2 h. Protein bands were visualized using an ECL chemiluminescence detection kit (32109; Thermo Fisher Scientific, Inc.) and a gel imaging system (ChemiDoc MP; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Absorbance analysis was performed using Image J software (Image J 1.8.0; National Institutes of Health, Bethesda, MD, USA).
3
Western Blot Analysis of GLOD4 in Testicular Development
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Testicular tissues at different development stages were homogenized and lysed using a radioimmunoprecipitation assay (RIPA) protein extraction kit (Solarbio, Beijing, China) and phenylmethanesulfonyl fluoride (PMSF) (Solarbio, Beijing, China), according to operating instructions. Protein concentrations were quantified using a commercial bicinchoninic acid (BCA) protein assay (Beyotime, Shanghai, China). Protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then were transferred onto polyvinylidene difluoride (PVDF) blotting membranes (Beyotime, Shanghai, China). The membranes were blocked with phosphate buffered saline tween-20 (PBST) containing 5% non-fat milk for 2 h at room temperature, and then incubated with either rabbit anti-GLOD4 polyclonal antibody (1:500; Bioss, Beijing, China) or anti-beta-actin polyclonal antibody (1:1000; Bioss, Beijing, China) at 4 °C overnight. After being washed with PBST, the membranes were incubated with goat anti-rabbit IgG/HRP antibody (1:5000; Bioss, Beijing, China) for 2 h at 37 °C. After being washed with PBST, the protein signals were visualized using NcmECL Ultra reagents (New Cell & Molecular Biotech Co. LTD, Suzhou, China) in an X-ray room. This experiment was biologically repeated eight times. Each biological replicate consisted of two technical replicates.
4
Western Blot Analysis of Cellular Proteins
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Cellular proteins were extracted in RIPA lysis buffer (BioTeke Corporation) on ice. An equal protein content of cell lysates was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrophoretically resolved, and then transferred onto polyvinylidene difluoride western blot membranes (Roche, Basel, Switzerland). The membranes were blocked for 3 h at 25 °C in 5% skim milk, and then incubated with specific primary and secondary antibodies. Immunoblots were detected using an ECL Western Blotting Substrate (Solarbio Science & Technology) and visualized using a Tanon 5200 digital imaging system (Tanon Science & Technology, Shanghai, China). Primary antibodies were caspase-3, cleaved-caspase-3, Bcl-2, Bax, GRP78, CHOP, IRE1, elF2α, ATF4, ATF6, p-38, p-p38, ERK, p-ERK, JNK, p-JNK, AKT and p-AKT (Wanleibio, Shenyang, China), β-actin, MPST, p-IRE1 (Bioss, Beijing, China), CBS, CSE (Omnimabs, Alhambra, CA, USA), and p-elF2α (Abbkine, Wuhan, China). Secondary antibodies (goat anti-rabbit IgG/HRP antibody, goat anti-mouse IgG/HRP antibody) were purchased from Bioss. Western blotting quantification results were evaluated with Image J software.
5
Western Blot Analysis of Protein Expression
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After transfection of SCs, they were homogenized and lysed using a radioimmunoprecipitation assay (RIPA) protein extraction kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Protein concentrations were then quantified using a commercial bicinchoninic acid (BCA) protein assay (Solarbio, Beijing, China). Next, the extracted proteins were denatured with 4x protein loading buffer (DTT, Solarbio, Beijing, China), resolved using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) blotting membranes (Beyotime, Shanghai, China). Membranes were first blocked with phosphate buffered saline tween-20 (PBST) containing 5% non-fat milk for 2 h at room temperature, followed by incubation with either rabbit anti-TPI1 polyclonal antibody (1:600; Bioss, Beijing, China) or anti-beta-actin polyclonal antibody (1:600; Bioss, Beijing, China) at 4°C overnight. On the next day, membranes were washed with PBST, and then incubated with goat anti-rabbit IgG/HRP antibody (1:5000; Bioss, Beijing, China) for 2 h at 37°C. Finally, they were washed with PBST and the protein signals were visualized using NcmECL Ultra reagents (New Cell & Molecular Biotech Co.LTD, Suzhou, China) in an X-ray room.
6
Western Blot Analysis of Protein Expression
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Total protein was extracted from each sample using a RIPA protein extraction kit (Solarbio Bio-Technology Co., Ltd, Beijing, China) according to the kit manual. Denatured protein samples of equal volume were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride blotting membranes (Beyotime Biotechnology, Shanghai, China). Transmembrane transfer was incubated with a rabbit anti-RTN4RL1 polyclonal antibody (1 : 500, Bioss Biotechnology Co., Ltd, Beijing, China), anti-TLR2 polyclonal antibody (1 : 500, Bioss Biotechnology Co., Ltd) and anti-β-actin polyclonal antibody (loading control) (1 : 1500, Bioss Biotechnology Co., Ltd) at 4°C overnight, respectively. After washing in PBST, membranes were incubated with goat anti-rabbit IgG/HRP antibody (1 : 5000, Bioss Biotechnology Co., Ltd). For more detailed information about the operational methods, see our previous report [32 (link)].
7
Anticancer Activity of SAL Compound
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SAL (purity, >99%) was purchased from Chengdu Ruifensi Biotechnology Co., Ltd. RPMI 1640 culture medium was purchased from HyClone (GE Healthcare Life Sciences), while fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd., and penicillin-streptomycin was purchased from Beyotime Institute of Biotechnology. Cell Counting Kit (CCK)-8 assay kit was purchased from Dojindo Molecular Technologies Inc., while DEX (Chinese medicine standard, H41020036) was purchased from Shanghai Shyndec Pharmaceutical Co., Ltd., and the cell cycle detection kit was purchased from Nanjing KeyGen Biotech Co., Ltd., and the Annexin V-FITC/PI apoptosis kit was purchased from BD Biosciences. The total RNA extraction kit was purchased from Tiangen Biotech Co., Ltd., while the reverse transcription and quantitative PCR (qPCR) kits were purchased from Toyobo Life Science, and the acridine orange stain was purchased from Biotopped Life Sciences. The rabbit anti-human c-Myc and GAPDH antibodies were purchased from ProteinTech Group, Inc., while the rabbit anti-human LC3A/B, Bax, BCL-2 and cleaved PARP antibodies were purchased from Cell Signaling Technology, Inc., and the goat anti-rabbit IgG-HRP antibody was purchased from BIOSS. Lastly, the PCR primers were synthesized by Shanghai Shenggong Biology Engineering Technology Service, Ltd.
8
Western Blot Analysis of Testis Proteins
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The proteins were extracted from the testis similarly as defined earlier [22 (link)]. The protein concentration was estimated by using a protein assay kit (Beyotime, Shanghai, China). Firstly, proteins were fixed in 12% Tricine SDS PAGE for gel electrophoresis and then transferred onto PVDF membranes (Roche, Indianapolis, IN, USA), after blocking in phosphate buffered saline tween 20 containing 5% nonfat milk, then membranes were incubated at 4 °C for whole night with anti SCP3 and anti SYCE3 antibody (1:1000, Abcam, Cambridge, UK) and anti β-actin (1:1000; Abcam, Cambridge, UK). After washing the membranes were incubated with goat anti rabbit IgG/HRP antibody (1:5000, Bioss, Beijing, China) for 1 hour, and finally bands intensity was determined by using ECL detection system (Pierce, Appleton, WI, USA).
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