Tandem affinity purifications were performed as previously described (15 (link), 61 (link)). Affinity purifications were performed as biological replicates and cell lines expressing SH-tagged GFP were used as negative controls. In brief, cell lines were incubated with 1–2 μg/ml doxycycline for 7–24 h to induce expression of SH-tagged bait proteins. Whole cell extracts were prepared in 50 mm HEPES (pH 8.0), 150 mm NaCl, 5 mm EDTA, 0.5% Nonidet P-40, 50 mm NaF, 1 mm Na3VO4, 1 mm PMSF, and protease inhibitor mixture. Cell lysates were cleared by centrifugation (13000 rpm, 20 min, 4 °C). Proteins were quantitated by Bradford assay using γ-globin as standard (Bio-Rad). 50 mg total lysate were incubated with StrepTactin Sepharose beads (IBA, Göttingen, Germany). Tagged proteins were eluted with d -biotin (Alfa-Aesar, Ward Hill, MA) followed by a second purification step using HA-agarose beads (Sigma-Aldrich). Protein complexes were eluted with 100 mm formic acid and immediately neutralized with triethylammonium bicarbonate buffer (Sigma-Aldrich). Samples were digested with trypsin (Promega, Fitchburg, WI), and the resultant peptides desalted and concentrated with customized reversed-phase tips (62 (link)). The volume of the eluted samples was reduced to ∼2 μl in a vacuum centrifuge and reconstituted with 5% formic acid.
γ globin
Manufactured by Bio-Rad
Sourced in United States
The γ-globin is a protein component of hemoglobin, the oxygen-carrying molecule in red blood cells. It is responsible for the fetal form of hemoglobin, which has a higher affinity for oxygen compared to the adult form. The γ-globin is an essential laboratory equipment used in various research and diagnostic applications related to hemoglobin and red blood cell function.
Automatically generated - may contain errors
Lab products found in correlation
Ecl western blotting system, by Thermo Fisher Scientific (3 mentions)
Protran ba85, by GE Healthcare (3 mentions)
Triethylammonium bicarbonate buffer, by Merck Group (2 mentions)
D biotin, by Thermo Fisher Scientific (2 mentions)
Ha agarose beads, by Merck Group (2 mentions)
Trypsin, by Promega (2 mentions)
Odyssey infrared imager, by LI COR (2 mentions)
Protease inhibitor, by Roche (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Edta free protease inhibitor, by Roche (1 mentions)
α flag, by Merck Group (1 mentions)
α akt, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
5 protocols using γ globin
1
Tandem Affinity Purification of Protein Complexes
2
Tandem Affinity Purification of Protein Complexes
3
Protein Extraction and Western Blot Analysis
4
Protein Isolation and Western Blot Analysis
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Cells were lysed using Nonidet-40 lysis buffer (50 mm HEPES (pH 7.4), 250 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 10 mm NaF, 1 mm Na3VO4, one tablet of EDTA-free protease inhibitor (Roche, Indianapolis, IN, USA) per 50 ml) or IP lysis buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 50 mm NaF, 1 mm Na3VO4, 1 mm PMSF, 5 μg/ml TPCK and protease inhibitor mixture) for 10 min on ice. Lysates were cleared by centrifugation (13000 rpm, 10 min, 4 °C). The proteins were quantified with BCA (Pierce, Grand Island, NY) or Bradford assay using γ-globin as a standard (Bio-Rad, Hercules, CA). Cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes Protran BA 85 (GE Healthcare, Little Chalfont, UK). The membranes were immunoblotted with the indicated antibodies. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies using the ECL Western blotting system (Thermo Scientific, Waltham, MA) or Odyssey Infrared Imager (LI-COR, Lincoln, NE).
5
Western Blot Analysis of SLC16A Transporters
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Cells were centrifuged, washed once with cold PBS and subsequently lysed in Nonidet-40 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM PMSF and one tablet of Roche EDTA-free protease inhibitor cocktail (Sigma-Aldrich) per 50 mL) for 10 min on ice. Lysates were then centrifuged (13,000 rpm, 10 min, 4 °C) and proteins were quantified with the Bradford assay using γ-globin as a standard (Bio Rad). Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes Protran BA85 (GE Healthcare). The membranes were incubated with α-SLC16A1 (Santa Cruz Biotechnology; sc-365501 mouse mAb 1:500 dilution), α-SLC16A3 (Santa Cruz Biotechnology; sc-376140 mouse mAb 1:500), α-FLAG (Sigma-Aldrich; F1804 mouse mAb 1:1000), α-HSP90 (BD Biosciences; 610418 purified mouse Ab 1:2000) or α-AKT (Cell Signaling Technologies; #4685 rabbit mAb 1:1000) and visualized with goat-anti-mouse IgG (115-035-003) or goat-anti-rabbit IgG (111-035-003) horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch; 1:5000) utilizing the ECL western blotting system (Thermo Fisher Scientific).
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