To best optimize the LAP purification procedure and minimize the possibility of carryover, cell culture, preparation of extracts and tandem affinity purification were standardized as described32 (link). Briefly, stable LAP cell lines were harvested using detergent. Lysates were clarified by centrifugation (43,000 rpm) and subjected to anti-GFP immunoprecipitation. Bound proteins were eluted from antibody beads using TEV protease, recaptured on S-protein agarose (Novagen), and eluted in 2x NuPAGE sample buffer (Invitrogen). Each purified set of interacting proteins was separated on an individual Bolt™ 4-12% Bis-Tris Plus Protein Gels (Invitrogen) and stained with Coomassie brilliant blue. Samples were run into gels for 20-40mm and divided into 20-40x1mm slices. Each excised lane was, reduced, carboxyamidomethylated and digested with trypsin, and mass spectrometry and peptide identification were carried out at the Stanford MS core facility as previously described145 (link).
Bolt 4 12 bis tris plus protein gels
Manufactured by Thermo Fisher Scientific
Bolt™ 4-12% Bis-Tris Plus Protein Gels are pre-cast polyacrylamide gels used for electrophoretic separation of proteins. They feature a Bis-Tris buffer system and a gradient concentration of 4% to 12% acrylamide.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Mes sds buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
2 protocols using bolt 4 12 bis tris plus protein gels
1
Optimized LAP Purification Protocol
2
Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Whole livers were
collected in gentleMACS
M tubes containing 2.5 mL of DMEM and homogenized using gentleMACS
dissociator (Miltenyi Biotec) RNA_01.01 program. Samples were mixed
with RIPA buffer (Thermo) supplemented with Halt protease inhibitor
cocktail (Thermo) for further lysis. Total protein concentration was
determined with by BCA protein assay (Thermo). Samples were mixed
with 4× Licor protein loading buffer (LiCor) denatured for 7
min at 95 °C and ran in Bolt 4–12% bis-Tris plus protein
gels (Invitrogen). The gels were ran with MES SDS buffer (Thermo)
at 200 V for 30 min. Proteins were transferred for 1 h to nitrocellulose
membranes at 20 V. Membranes were blocked overnight in 5% bovine serum
albumin at 4 °C. Primary antibodies against β-actin (Cell
Signaling, 4970S, 1:1000) and B4galnt2 (Novus, NBP1-91229, 1:1000)
were incubated overnight at 4 °C in TBST (0.1% Tween 20). Membranes
were washed with TBST and incubated with anti-rabbit secondary antibody
(LI-COR, 926-68073, 1:5000) in TBST for 1 h at room temperature. Proteins
were visualized using an Odyssey infrared imaging system.
collected in gentleMACS
M tubes containing 2.5 mL of DMEM and homogenized using gentleMACS
dissociator (Miltenyi Biotec) RNA_01.01 program. Samples were mixed
with RIPA buffer (Thermo) supplemented with Halt protease inhibitor
cocktail (Thermo) for further lysis. Total protein concentration was
determined with by BCA protein assay (Thermo). Samples were mixed
with 4× Licor protein loading buffer (LiCor) denatured for 7
min at 95 °C and ran in Bolt 4–12% bis-Tris plus protein
gels (Invitrogen). The gels were ran with MES SDS buffer (Thermo)
at 200 V for 30 min. Proteins were transferred for 1 h to nitrocellulose
membranes at 20 V. Membranes were blocked overnight in 5% bovine serum
albumin at 4 °C. Primary antibodies against β-actin (Cell
Signaling, 4970S, 1:1000) and B4galnt2 (Novus, NBP1-91229, 1:1000)
were incubated overnight at 4 °C in TBST (0.1% Tween 20). Membranes
were washed with TBST and incubated with anti-rabbit secondary antibody
(LI-COR, 926-68073, 1:5000) in TBST for 1 h at room temperature. Proteins
were visualized using an Odyssey infrared imaging system.
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