In vitro phosphorylation assays with purified MARK2 or MARK3 were carried out in buffer containing 10 mM MOPS pH 7.2, 50 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.2 mM CaCl2, 1xPhosStop (Roche), 0.2 mM ATP for 1.5h at room temperature. In vitro phosphorylation assays with purified MLCK were carried out in buffer containing 10 mM MOPS pH 7.2, 50 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 1xPhosStop (Roche), 0.2 mM ATP, and 0.1 μM calmodulin for 1.5h at room temperature. Control reactions lacked the kinase. Final protein and peptide concentrations in the assays were as follows: MYPT1: (fragment 654-880, Millipore): 1 μM; His-MRLC: 2 μM (prepared as described in 93 (link); MARK2 (SignalChem): 0.4-10 μg/ml; MARK3 (SignalChem): 0.4-10 μg/ml; MLCK (SignalChem): 4 μg/ml; ROCK2 (SignalChem): 0.4-4 μg/ml; CHKtide (SignalChem): 0.1 mg/ml; MRLC peptide (SignalChem): 0.4 mg/ml. The phosphorylation reaction was stopped by either flash freezing the samples in liquid nitrogen followed by the addition of SDS loading dye and boiling of the sample or the addition of 5% acetonitrile and 0.05% trifluoroacetic acid. The extent of phosphorylation was determined by western blot analysis or intact protein HPLC-mass spectrometry as described before94 .
1xphosstop
Manufactured by Roche
Sourced in Germany
1xPhosStop is a phosphatase inhibitor cocktail designed for the inhibition of serine/threonine and tyrosine phosphatases during sample preparation and protein extraction. It is a ready-to-use solution that can be added directly to the sample.
Automatically generated - may contain errors
Lab products found in correlation
Rock2, by Sino Biological (1 mentions)
Mark3, by Sino Biological (1 mentions)
Chktide, by Sino Biological (1 mentions)
1xprotease inhibitors cocktail, by Roche (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Anti rorα, by Abcam (1 mentions)
Anti rabbit irdye 680rd, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
4 protocols using 1xphosstop
1
In vitro kinase assays for MARK, MLCK
2
Western Blot Protein Quantification
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For Western blotting, following appropriate treatment where applicable, cells were lysed in Hepes lysis buffer [100 mmol/l NaCl, 1% (v/v) Triton‐X‐100, 0.5% (w/v) sodium deoxycholate, 0.2% (w/v) SDS, 2 mmol/l Na2EDTA, 10 mmol/l Hepes buffer (pH 7.5), 1xPhosSTOP (cocktail of phosphatase inhibitors, Roche) and 1xProtease inhibitors cocktail (Roche, Mannheim, Germany). Protein concentration was determined by the Bradford colorimetric assay (Pierce, Thermo Fisher Scientific Waltham, MA, USA). Immunoblot analysis of cell lysates was performed as previously described 30 . To normalize protein load amounts, the blots were stripped (Re‐Blot Plus Mild Solution, Cat. No: 2502, Millipore, Thermo Fisher Scientific Waltham, MA, USA) and re‐probed with anti‐β‐tubulin monoclonal antibody (Cat. No: T4026; Sigma‐Aldrich).
3
Adipose Tissue Protein Extraction and Analysis
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Protein extraction from adipose tissue was previously described (Lau et al., 2015 (link)) with modifications. Inguinal white adipose tissues were homogenized in 1 mM EDTA, 10 mM Tris, and 0.25 M sucrose (pH 7.5) with 1xComplete protease inhibitor and 1xPHOS-STOP (Roche Diagnostics, Mannheim, Germany). Infranatant and pellet were separated from the top layer of fat cake after centrifugation. Detergent was then added to a final concentration of 1% Triton X-100, 1% NP-40, and 0.1% SDS for the infranatant (cytosolic proteins) and pellet (nuclear and membrane proteins) separately, incubated for 30 min and sheared eight times with an insulin syringe. The pellet was sheared multiple times using P200 pipette tips and insulin syringe. Protein concentration was measured with BCA reagent (Thermoscientific; Pierce, Rockford, IL). Immunoblot analysis was performed as described previously except that the membranes were blocked in 5% skim milk and probed with anti-RORα (ab60134 1:1000; Abcam), anti-AKT (#9272, 1:1000; Cell Signaling Technology, Danvers, MA), pAKT (ser473) (#4058; 1:1000; Cell Signaling Technology), anti-TBP (sc-204; 1:1000, Santa Cruz Biotechnology), or anti-α-tubulin (#2144, 1:1000; Cell Signaling Technology).
4
Western Blot Analysis of NF1 Protein
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Cells were washed twice with chilled PBS and lysed with RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl (Merck), 1mM EDTA (Sigma), 0.5% Igepal CA-630 (Sigma)) supplemented with 3mM DTT (Thermo Fisher), 1mM PMSF (Sigma), 1mM sodium orthovanadate (Sigma), 5mM NaF (Sigma), 10 ug/ml leupeptin (Sigma), 5ug/ml aprotinin (Sigma) and 1xPhosSTOP (Roche). Lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Lysates were boiled with 1X Laemmli buffer (50% glycerol (v/v) (Sigma), 10% SDS (m/v) (Merck) , 0.05% bromophenol blue (m/v) (Sigma), 25% Tris HCl 1M pH 6.8 (v/v) (Millipore), 5% betamercaptoethanol (v/v) (Sigma) in distilled water) and 90 mg of protein was subjected to SDS-PAGE and transferred onto PVDF membranes (18 hours 90 mA at 4 C). Membranes were blocked with Odyssey Blocking Buffer (TBS, LI-COR) and incubated with rabbit anti-NF1 Antibody (Bethyl laboratories) at 4 C overnight; and with mouse anti-atubulin (Sigma) 1 hour at room temperature. Membranes were then incubated with IRDye 680RD anti-Rabbit and IRDye 800CW anti-Mouse secondary antibodies (1:10,000 each, LI-COR) for 1 hour at room temperature and scanned with the Odyssey CLx (LI-COR) using the Image Studio Lite (LI-COR).
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