Co-immunoprecipitations were conducted according to the protocol in [77 (link)]. Briefly, the protein concentration of E−Du145 and E−PC3 lysates were determined by Lowry assay (Biorad Dc assay cat: 500, Biorad, UK). Aliquots of cell lysates containing 500 μg protein were transferred to new tubes and the volume adjusted to 400 μL with lysis buffer. 30 μL packed Pierce Protein A/G Plus Agarose Beads (Life Technologies #20423) were combined with 500 μg whole cell lysate on a rocker at 4 °C for 1 h to clear the lysate. 500 μg cleared protein lysate and 5 μg/mL primary antibody were rocked overnight at 4 °C. Control samples had 5 μg isotype-matched control IgG added. 30 μL of fresh packed beads were added to the protein lysate with primary antibody and rocked at 4 °C for 1 h. Samples were centrifuged for 5 min at 10,000 × g. The beads were washed three times with 500 μL immunoprecipitation buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, 1% Tween) before applying 25 μL 2× SDS sample buffer (Bio-Rad 161–0737) with 10% β-mercaptoethanol (Sigma-Aldrich M6250) and boiled at 100 °C for 5 min. The samples were centrifuged briefly at room temperature before loading with Prot/Elec tips (Bio-Rad 2239916) on a western blot.
Prot elec tips
Manufactured by Bio-Rad
The Prot/Elec Tips are disposable pipette tips designed for use in protein electrophoresis applications. They are made of high-quality materials to ensure accurate and consistent sample delivery.
Automatically generated - may contain errors
3 protocols using prot elec tips
1
Co-immunoprecipitation Workflow for Protein Interactions
2
Plasma Extraction from Fish Blood
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Blood was collected using a modified tail ablation method. Briefly, fish was euthanized in ice-water and its surface was wiped dry. Tail was removed by a cut at the end of anal fin and the fish was held with the wound facing down. Whole blood was collected at the dorsal aorta using P20 micropipette fitted to an elongated tip (Prot/Elec Tips, Bio-Rad) and aspired into pre-chilled Eppendorf tubes. Both pipette tips and tubes were pre-coated with EDTA by submerging in 18 mg/mL EDTA solution for 24 hrs and then dried prior to use. Plasma was obtained as clear supernatant after centrifugation at 1000 g for 10 min at 4 °C. Typical yield of whole blood is 5–10 μl from male fish and 8–15 μL from female fish. Whole blood was collected to pre-chilled tubes on ice and then centrifuged within 10 min to obtain plasma. For MS analyses, each plasma sample was pooled from 9 fish. Protein concentration was determined using RCDC protein assay kit (Bio-Rad) according to the manufacturer’s protocol. For quick evaluation of protein abundance patterns, six individual plasma samples (3 males and 3 females, 0.5 μL each) were analyzed using 10% SDS gel followed by silver staining.
3
Assessing Liver Function in Fish
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Bilirubin assay kit and alanine aminotransferase (ALT) activity assay kit (Sigma) were used to assess functional status of fish liver. For bilirubin assay, only plasma was used. For ALT activity assay, both plasma and liver protein extracts were used.
Whole blood was collected at the dorsal aorta after tail ablation using P20 micropipettor fitted to an elongated tip (Prot/Elec Tips, Bio-Rad) and aspired into pre-chilled Eppendorf tubes. Both pipette tips and tubes were pre-coated with EDTA (submerging in 18 mg/mL EDTA solution for one day and then dried). Blood of 3–6 fish were pooled as one sample. Plasma was obtained as clear supernatant after centrifugation at 1000 g for 10 min at 4°C and stored at -80°C. Bilirubin assay was completed within 2 days of plasma collection.
To extract total protein, liver tissue was homogenized in ALT assay buffer (1:15 w/v) on ice and then centrifuged at 14,000 rpm at 4°C for 10 min. Supernatant was collected. Protein concentration was estimated based on Bradford method using Bio-Rad Protein Assay Dye Reagent and Bovine Serum Albumin standard (Sigma). ALT assay was performed following manufacturer’s protocol, while bilirubin assay was scaled down after confirming the linearity of bilirubin standard (S1 Fig ).
Whole blood was collected at the dorsal aorta after tail ablation using P20 micropipettor fitted to an elongated tip (Prot/Elec Tips, Bio-Rad) and aspired into pre-chilled Eppendorf tubes. Both pipette tips and tubes were pre-coated with EDTA (submerging in 18 mg/mL EDTA solution for one day and then dried). Blood of 3–6 fish were pooled as one sample. Plasma was obtained as clear supernatant after centrifugation at 1000 g for 10 min at 4°C and stored at -80°C. Bilirubin assay was completed within 2 days of plasma collection.
To extract total protein, liver tissue was homogenized in ALT assay buffer (1:15 w/v) on ice and then centrifuged at 14,000 rpm at 4°C for 10 min. Supernatant was collected. Protein concentration was estimated based on Bradford method using Bio-Rad Protein Assay Dye Reagent and Bovine Serum Albumin standard (Sigma). ALT assay was performed following manufacturer’s protocol, while bilirubin assay was scaled down after confirming the linearity of bilirubin standard (
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