Sperm‐cell lysates, SEs‐depleted seminal plasma, and SEs‐protein concentrations were determined using a BCA protein assay kit (P0010S; Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, 30 μg of total protein was heated to 95 °C for 10 min in 1 × DTT‐containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDS‐polyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene fluoride membranes (BioTrace; Bio‐Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti‐CD81, anti‐HSP‐70, anti‐ALIX, anti‐PHGDH, anti‐LGALS3BP, anti‐SEMG1 (Abclonal, Woburn, MA, USA), anti‐ACTB, anti‐GAPDH (Ray Antibody, Beijing, China), and anti‐Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each specific horseradish peroxidase‐conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS‐Tween solution (TBS‐T) five times for 25 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).
Anti cd81
Manufactured by ABclonal
Sourced in United States
Anti-CD81 is a laboratory equipment product. It is a monoclonal antibody that specifically binds to the CD81 protein. CD81 is a cell surface protein that plays a role in various cellular processes. The primary function of Anti-CD81 is to detect and quantify the presence of CD81 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Biotrace, by Bio-Rad (5 mentions)
Anti calnexin, by Bioworld Technology (5 mentions)
Anti cd63, by ABclonal (5 mentions)
Anti p akt anti alix, by ABclonal (4 mentions)
Anti hmgb1, by ABclonal (4 mentions)
Anti tsg101, by ABclonal (4 mentions)
Anti caspase 3, by ABclonal (4 mentions)
Anti bcl 2, by ABclonal (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Anti akt, by ABclonal (4 mentions)
6 protocols using anti cd81
1
Seminal Plasma Protein Characterization
2
Protein Quantification and Western Blot Analysis
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The concentrations of all the protein we extracted from cells , exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT-containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDSpolyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene uoride membranes (BioTrace; Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti-CD63,anti-CD81, anti-TSG101,anti-AKT,anti-p-AKT anti-ALIX, anti-HMGB1, anti-Caspase-3,anti-Bcl2 (Abclonal, Woburn, MA, USA), anti-ACTB, anti-GAPDH (Ray Antibody, Beijing, China), and anti-Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each speci c horseradish peroxidase-conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS-Tween solution (TBS-T) three times for 30 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).
3
Protein Quantification and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The concentrations of all the protein we extracted from cells , exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT-containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDSpolyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene uoride membranes (BioTrace; Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti-CD63,anti-CD81, anti-TSG101,anti-AKT,anti-p-AKT anti-ALIX, anti-HMGB1, anti-Caspase-3,anti-Bcl2 (Abclonal, Woburn, MA, USA), anti-ACTB, anti-GAPDH (Ray Antibody, Beijing, China), and anti-Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each speci c horseradish peroxidase-conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS-Tween solution (TBS-T) three times for 30 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).
4
Protein Quantification and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentrations of all the protein we extracted from cells , exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT-containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDSpolyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene uoride membranes (BioTrace; Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti-CD63,anti-CD81, anti-TSG101,anti-AKT,anti-p-AKT anti-ALIX, anti-HMGB1, anti-Caspase-3,anti-Bcl2 (Abclonal, Woburn, MA, USA), anti-ACTB, anti-GAPDH (Ray Antibody, Beijing, China), and anti-Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each speci c horseradish peroxidase-conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS-Tween solution (TBS-T) three times for 30 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).
5
Protein Quantification and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentrations of all the protein we extracted from cells , exosomes and tissues were determined using a BCA protein assay kit ( Beyotime Biotech, Jiangsu, China) according to manufacturer instructions. Subsequently, a certain amount of total protein[20ug-80ug] was heated to 95 °C for 10 min in 1 × DTT-containing sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDSpolyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene uoride membranes (BioTrace; Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% bovine serum albumin for 1 h, and polyclonal antibodies, including anti-CD63,anti-CD81, anti-TSG101,anti-AKT,anti-p-AKT anti-ALIX, anti-HMGB1, anti-Caspase-3,anti-Bcl2 (Abclonal, Woburn, MA, USA), anti-ACTB, anti-GAPDH (Ray Antibody, Beijing, China), and anti-Calnexin (Bioworld, Dublin, OH, USA), were used for immunoblotting at 4 °C overnight. Each speci c horseradish peroxidase-conjugated secondary antibody (Ray Antibody) was added accordingly after the membranes were washed in TBS-Tween solution (TBS-T) three times for 30 min, and signals were detected by enhanced chemiluminescence (Pierce, Rockford, IL, USA).
6
Quantitative Analysis of miR-4787-3p Expression
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The protein was analyzed by western blot as previously described [22] (link). The primary antibodies used in this study were as follows: anti-CD81(ABclonal, Wuhan, China), anti-CD63(ABclonal, Wuhan, China).
Quantitative RT-PCR (qRT-PCR) qRT-PCR was performed to assess the expressions of miR-4787-3p. U6 was used as internal controls. The primers were shown as follows: miR-4787-3p: CACTGCCCCGCGCAAA; U6: TCGTAAGCGTTCCATATTTTTAA. The conditions of the ampli cation reaction were as follows: 95°C for 10 mine, 95°C for 10 s, 40 cycles of 66°C. The relative expression of miR-4787-3p was estimated by 2 - ΔCT .
Quantitative RT-PCR (qRT-PCR) qRT-PCR was performed to assess the expressions of miR-4787-3p. U6 was used as internal controls. The primers were shown as follows: miR-4787-3p: CACTGCCCCGCGCAAA; U6: TCGTAAGCGTTCCATATTTTTAA. The conditions of the ampli cation reaction were as follows: 95°C for 10 mine, 95°C for 10 s, 40 cycles of 66°C. The relative expression of miR-4787-3p was estimated by 2 - ΔCT .
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