Myc-Kv4.2 (co-transfected with p38) was expressed in HEK293T cells and purified with myc-magnetic beads (Thermo Scientific, #88842). Kv4.2 Phosphorylation at T602 and T607 after purification was measured by saturated phospho-specific and total Kv4.2 antibody pulldown combined with western blot. About 2/3 of purified Myc-Kv4.2 was phosphorylated at T607 and was used for proteolysis. Equal amount of Myc-Kv4.2 were then incubated with 100 ng of either GST, GST-Pin1, and GST-Pin1C113S in a buffer containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MgCl2, supplemented with phosphatase inhibitors. After 30 min incubation at room temperature, reaction mixtures were cooled on ice, and subtilisin (Sigma-Aldrich, P5380) was added for a further 1 min on ice. The reaction was stopped by the addition of boiling sample buffer, and the proteolytic fragments were resolved by 4–12% SDS-PAGE and visualized by western blot analysis.
Myc magnetic beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Myc-magnetic beads are a versatile tool used in various biotechnology applications. These beads are coated with an anti-Myc antibody, which allows for the efficient capture and purification of Myc-tagged proteins from complex samples. The magnetic properties of the beads facilitate easy separation and washing steps, making them a convenient choice for researchers working with Myc-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Subtilisin, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Bi d1870, by Selleck Chemicals (1 mentions)
Anti phosphoserine, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Roche (1 mentions)
U0126, by Selleck Chemicals (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Sab1305535 40tst, by Merck Group (1 mentions)
5 protocols using myc magnetic beads
1
Kv4.2 Phosphorylation and Proteolysis
2
Antibody-mediated Signaling Pathway Analysis
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Primary antibodies used in this study were as follows: anti-phospho REPS1 S709 (12005), anti-REPS1 (6404), anti-phospho Akt S473 (4060), anti-Akt (4691), anti-phospho p70 S6K (9234), anti-p70 S6K (2708), anti-phospho threonine (9386), anti-phospho tyrosine (9416), anti-phospho ERK T202/T204 (4370), anti-ERK1/2 (4695), anti-phospho RSK S380 (11989), anti-RSK1/2/3 (9355), anti-RSK1 (8408), anti-RSK2 (5528), anti-Myc (2278) were purchased from Cell Signaling Technology (Danvers, MA, USA). anti-phospho serine (61-8100, Invitrogen, Carlsbad, CA, USA), anti-RSK3 (sc-1431, Santa Cruz, Dallas, Texas, USA), anti-GAPDH (LF-PA0212, AB frontier, Seoul, South Korea), anti-Flag-M2 (F1804, Sigma Aldrich, Kenilworth, NJ, USA), and anti-HA (11 867 423 001, Roche, Basel, Switzerland). Flag magnetic beads (A36798) and Myc magnetic beads (88842) were purchased from Thermo Fisher (Waltham, MA, USA). GDC0349 (S8040, Selleckchem, Houston, TX, USA), rapamycin (37094, Sigma Aldrich, Kenilworth, NJ, USA), BI-D1870 (S2843, Selleckchem), and U0126 (1144, Selleckchem) were additionally used for experiments in this study. Alexa-555 EGF (E35350) and Alexa-555 Transferrin (T35352) were purchased from Thermo Fisher.
3
Antibody Validation and Cell Signaling Assays
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Flag tag antibody (Cat. #AE063), HA tag antibody (Cat. #AE008), phospho-STMN1-S25 antibody (Cat. #AP0220), MYC tag antibody (Cat. # AE070) and ubiquitin antibody (Cat. #A19686) were purchased from Abclonal. Beta-tubulin (Cat. #2146), stathmin1 antibody (Cat. #3352), acetylated lysine antibody (Cat. #9441), phospho-ERK1/2 antibody (Cat. #4370), ERK1/2 antibody (Cat. #4695) and actin antibody (Cat. #3700) were purchased from Cell Signaling Technology. TSA (Cat. #HY-15144), NAM (Cat. # HY-B0150), MG132 (Cat. # HY-13259) and Protein A/G (Cat. # HY-K0202) were purchased form MedChemExpress. CHX (Cat. # S7418) was purchased from Selleck. Flag agarose beads (Cat. #B23102) was purchased from Bimake. HA magnetic beads (Cat. #88836) and MYC magnetic beads (Cat. #88842) were purchased from ThermoFisher Scientific. Binimetinib (Cat. #T2508) was purchase form Topscience.
4
Co-Immunoprecipitation of Transfected Proteins
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For Co-IP analysis, HEK 293T cells grown to 60% confluency were transfected with a total of 10 μg of the indicated plasmids. At 24 h post-transfection, the medium was carefully removed, and the cells were washed with ice-cold PBS. Then the cells were lysed in 1 mL RIPA buffer at 4°C on a horizontal shaker for 1 h. Cells lysates were centrifugated at 16000 × g at 4°C for 20 min, then the supernatant was incubated with anti-Flag (M8823; Sigma-Aldrich, St. Louis, MO, United States) or Myc magnetic beads (88842, Thermo Fisher Scientific, Waltham, MA, United States) overnight at 4°C. Finally, immunoprecipitates and total cell lysates (TCL) were analyzed by western blotting using the indicated antibodies.
5
Ubiquitin Enrichment and Detection
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Twelve-day-old ProUBQ:mCherry-CPK4-myc Col, ProUBQ:mCherry-CPK4-myc pub44, ProUBQ:mCherry-CPK4-myc ProUBQ:Venus-PUB44-Flag plants grown on 1/2 MS medium were treated with 50 mM MG132 for 4 h and ground to powder in liquid nitrogen. Total proteins were extracted with protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol, 50 mM MG132, and 1× protease inhibitor mix). The supernatants were centrifuged twice at 12,000 × g for 15 min and passed through 0.45 µm filter. The resulting supernatants were incubated with prewashed myc magnetic beads (Thermo Fisher) at 4 °C for overnight. The magnetic beads were washed 4 times with the 1 × extraction buffer and boiled with 1 × SDS loading buffer at 95 °C for 5 min. The proteins were separated on SDS-PAGE and detected with anti-myc (SAB1305535-40TST, Sigma, 1:5,000 dilution) and anti-Ub rabbit polyclonal antibody (10201-2-AP, Proteintech, 1:1,000 dilution).
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