Exosomes and cells were lysed with RIPA (Beyotime, China) extraction reagent plus protease inhibitor PMSF (Beyotime, China). Protein concentration was determined using the BCA protein assay kit from Beyotime Biotechnology. Total proteins were isolated by 10% sodium dodecyl‐sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a 0.22‐μm polyvinylidene difluoride membrane (Millipore, USA). The exosomal protein samples were incubated with anti‐TSG101 (Proteintech, USA), Calnexina (Proteintech, USA), and anti‐CD9 (Proteintech, USA) primary antibodies, washed with tris‐buffered saline Tween‐20 (TBST), and incubated with horseradish peroxidase‐labeled goat anti‐rabbit secondary antibodies (Beyotime, China); β‐actin (Beyotime, China) was used as an internal control. Protein bands were displayed using a fully automatic chemiluminescence imager.
Horseradish peroxidase labeled goat anti rabbit secondary antibody
Manufactured by Beyotime
Sourced in China
Horseradish peroxidase-labeled goat anti-rabbit secondary antibody is a protein used for detecting and quantifying the presence of rabbit primary antibodies in various laboratory techniques. It is a conjugate of a secondary antibody and the enzyme horseradish peroxidase, which catalyzes a colorimetric reaction for signal amplification and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Anti tsg101, by Proteintech (1 mentions)
Anti cd9, by Proteintech (1 mentions)
0.22 μm polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
β actin, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Gel purification kit, by Takara Bio (1 mentions)
Restriction endonuclease bamhi, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Annexin 5 pe 7aad, by Keygen Biotech (1 mentions)
Cck 8, by Beyotime (1 mentions)
Cell apoptosis detection kit, by Keygen Biotech (1 mentions)
Skimmed milk, by Beyotime (1 mentions)
Enhanced chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bca kit, by Merck Group (1 mentions)
6 protocols using horseradish peroxidase labeled goat anti rabbit secondary antibody
1
Exosomal Protein Characterization by Western Blot
2
Lymphocyte Separation and T-cell Apoptosis
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Human lymphocyte separation medium were obtained from Tianjin TBD Co. (Tianjin, China), and Jurkat leukemic T cells from human peripheral blood (Shanghai Institute of Cell Library, Shanghai, China). RPMI-1640 and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA) and Lipofectamine™ 2000 from Invitrogen-Life Technologies (Carlsbad, CA, USA). G418 and CCK-8 were obtained from Beyotime Institute of Biotechnology (Shanghai, China) and DMSO from Sigma (St. Louis, MO, USA). Annexin V-PE/7AAD and the cell apoptosis detection kit were obtained from Nanjing KeyGen Biotech (Nanjing, China) and the TRIzol reagent from Invitrogen-Life Technologies. The RNA extraction reagent was purchased from BioFlux, Hangzhou, China. The QF-PCR kit, HOXA5, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primer, restriction endonuclease BamHI, T4 DNA ligase, and gel purification kit were purchased from Takara (Shiga, Japan). siRNA sequence targeting HOXA5, and the negative control siRNA sequence were purchased from Adicon Co. (Shanghai, China). Rabbit anti-human HOXA5 polyclonal antibodies were purchased from Abcam (Cambridge, UK) and horseradish peroxidase-labeled goat anti-rabbit secondary antibodies were purchased from the Beyotime Institute of Biotechnology. Other reagents were developed and purified in China.
3
PSMA Immunohistochemistry of Xenograft Tissues
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PC-3 and C4-2 xenograft tissues were collected, fixed in paraformaldehyde, embedded in paraffin, and sectioned using a microtome. The immunohistochemistry procedure for tissue sections was then performed. After deparaffinization, sections were blocked in 3% H2O2 for at least 10 minutes. Antigen retrieval was performed in citric acid at high temperature and high pressure. Sections were incubated with a rabbit anti-human PSMA monoclonal antibody (Abcam) (1:400 dilution) at 4°C overnight. Next, sections were incubated with a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Beyotime Institute of Biotechnology) at room temperature for 1 hour. In all the procedures, sections were washed with PBS after each step. Finally, the results were developed with the DAB reagent, and cell nuclei were counterstained using hematoxylin. Sections were mounted using neutral balsam and observed under a light microscope.
4
IF-TSA Staining Protocol
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The protocols for IF-TSA staining [Figure 1b ] were the same as for standard IF staining [Figure 1a ] except for the following steps. Horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Beyotime, A0208, Jiangsu, China) was used to substitute for Alexa Fluor 488-labeled goat anti-rabbit secondary antibody, and then, the sections were incubated with fluorescein isothiocyanate (FITC)-labeled tyramide (1:50) in a ×1 amplification solution (Perkin Elmer, Wellesley, MA, USA) for 10 min at room temperature.
5
Western Blot Analysis of AKT/mTOR Signaling
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BMSCs were cultured in differentiating medium for 48 h. Cells (1×106) suspended in 0.5 ml of Laemmle buffer (50 mM tris pH6.8, 1.25% SDS, 10% glycerol) were lysed using SoniConvert sonicator (DocSense) and denatured by heating at 100°C for 15 min. Then, protein concentration was determined using BCA kit (Sigma-Aldrich; Merck KGaA). Total protein (20 µg) was separated in each lane via 6–12% gradient SDS-PAGE electrophoresis, transferred onto nitrocellulose membranes (EMD Millipore) and subsequently blocked with PBS containing 5% skimmed milk (Beyotime Institute of Biotechnology) at room temperature for 30 min. The membranes were incubated with primary antibodies against: AKT (1:3,000; cat. no. ab9905), AKT phosphor T308 (1:2,000; cat. no. ab38449), mTOR (1:1,000; cat. no. ab2732), mTOR phosphor S2448 (1:1,000; cat. no. ab109268) and β-actin (1:5,000; cat. no. ab8226; all from Abcam) for 1 h at room temperature. Membranes were washed three times with PBS containing 0.1% Tween-20 (Beyotime Institute of Biotechnology) and subsequently incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:5,000; cat. no. ab7090; Abcam) for 1 h at room temperature. Proteins bands were visualized on X-ray film using enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.) and quantified using ImageJ software (version-2.0; National Institutes of Health).
6
Quantitative Western Blot Analysis
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Western blot assays were performed as previously described. Cells were lysed (Bio-Rad), and the membranes were probed with rabbit anti-human-ILK monoclonal antibody (Abcam), rabbit anti-α-SMA monoclonal antibody (Abcam), rabbit anti-AKT monoclonal antibody (Abcam), rabbit anti-p-AKT monoclonal antibody (Abcam), and rabbit anti-GAPDH monoclonal antibody (Abcam) as an internal reference overnight at 4 °C. Membranes were washed in RIPA lysis buffer (Beyotime, CHN) and centrifuged at 12 000 g and 4 °C for 5 min to extract the total protein. The total protein concentration was measured using the bicinchoninic acid assay kit (Beyotime, Cnina) according to the manufacturer's instructions. We separated 20 nl of total protein in each sample hole using 10% SDS-PAGE and transferred to a nitrocellulose membrane. The proteins were then washed with TBST and incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Beyotime, CHN) for 1 h at 37 °C, and incubated with enhanced chemiluminescence (Thermo, USA) for 5 min in the dark at room temperature. The photographs were collected using a gel imaging system (Bio-Rad) and analyzed using Image Lab software (Bio-Rad).
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