For Western blot analysis of Gaf1-HA, proteins were extracted with trichloroacetic acid (TCA). For immunoprecipitations (IP) of Gaf1-HA, proteins were extracted with lysis buffer 1 (50 mM Tris-HCl [pH 7.5], 20% glycerol, 150 mM NaCl, 5 mM EDTA [pH 8.0], 1 mM dithiothreitol [DTT], 0.1% Triton X100, protease inhibitor cocktail, phosphatase inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride). Cells were broken for 15 min with glass beads and centrifuged for 10 min at 1,000 × g and the supernatant was collected. Approximately 2 mg of proteins was prepared and precleared with 20 µl of a mixture of protein A Sepharose and protein G Sepharose beads (GE Healthcare). Two microliters of hemagglutinin (HA) antibodies was added to the cleared extract and incubated overnight at 4°C, followed by addition of 20 µl of a mixture of protein A and G Sepharose beads. After 3 h of incubation, the beads were washed five times with lysis buffer 2 (50 mM Tris-HCl [pH 7.5], 20% glycerol, 150 mM NaCl, and 0.1% Triton X100). For phosphatase treatment, the beads were diluted with λ-phosphatase reaction buffer and 2 mM MnCl2 and then incubated with 200 U of λ-phosphatase (New England Biolabs) at 30°C for 30 min. The beads were then washed once with lysis buffer 2, resuspended in 25 µl 2× sample buffer, and boiled for 3 min.
λ phosphatase
Manufactured by New England Biolabs
Sourced in United States
λ-phosphatase is an enzyme that can remove phosphate groups from a variety of substrates, including proteins, nucleic acids, and small molecules. It is commonly used in research applications to study the role of phosphorylation in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Pmp buffer, by New England Biolabs (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (2 mentions)
Pierce crosslink ip kit, by Thermo Fisher Scientific (2 mentions)
Chemiluminescent reagent, by PerkinElmer (2 mentions)
Protean ief cell, by Bio-Rad (2 mentions)
Ipg strips with 4 7ph range, by Bio-Rad (2 mentions)
Phos tag page, by Fujifilm (2 mentions)
Las 3000 imaging system, by Fujifilm (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Phos tag reagent, by Fujifilm (2 mentions)
Phos tag, by Fujifilm (2 mentions)
Metallophosphatase buffer, by New England Biolabs (2 mentions)
Cenp a, by New England Biolabs (2 mentions)
Vectashield with dapi, by Vector Laboratories (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Image lab 5, by Bio-Rad (2 mentions)
83 protocols using λ phosphatase
1
Gaf1-HA Analysis by Western Blot and Immunoprecipitation
2
Phosphatase Regulation of CaMKII Phosphorylation
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To test how the presence of phosphatase affects the phosphorylation status of CaMKII when the kinase is active, increasing amounts of λ-phosphatase (200–800 units, New England Biolabs) and 1 mM MnCl2 was added to samples of glass-immobilized CaMKII, in the presence of activation buffer containing 5 μM Ca2+/CaM. 1 unit of λ-phosphatase is defined as the amount of enzyme that hydrolyzes 1 nmol of p-nitrophenyl phosphate in 1 min at 30°C (New England Biolabs). After 45 min of incubation, the flow chambers were washed with 2 mL DPBS to remove the λ-phosphatase and the activation buffer. The phosphorylation of Thr 286 and Thr 305/306 was then examined using the immunofluorescence assay described above. No further reduction in phosphorylation was observed upon adding more than 400 units of λ-phosphatase and this is considered as a saturating amount of phosphatase.
3
GST-BRCT Domain Pulldown Assay
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Bacterially expressed fusion proteins containing GST and the BRCT domains of MDC1 (residues 1893–2082) or GST alone immobilized onto Glutathion Sepharose 4B beads (GE Heathcare) and incubated with lysates prepared from HeLa cells or cells transiently transfected with GFP-ID3 expression vector for 3 h at 4 °C. Cells were lysed in TEN100 buffer (20 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 10 μg ml−1 leupeptin, and 10 μg ml−1 aprotinin). If λ-phosphatase treatment was required, TEN100 lysates were incubated with 400 units of λ-phosphatase (New England BioLabs) at 30 °C for 30 min. The GST beads were washed five times with NTEN buffer (0.5% NP-40. 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 300 mM NaCl), and bound proteins were separated by SDS–PAGE and analyzed by western blotting using the appropriate antibodies.
4
Gel Filtration Chromatography of Sae2 Variants
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The gel filtration chromatography was carried out using a Superose 6 10/300 GL column (GE Healthcare) on an ÄKTA prime system (GE Healthcare). The separation buffer contained 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, and 0.5 mM β-mercaptoethanol. The recombinant Sae2/pSae2 variant (~75 μg) was diluted into 200 μl of separation buffer, supplemented with 1 mM manganese chloride, and either treated with λ phosphatase (4'800 U, New England Biolabs) or mock-treated (without λ phosphatase) for 90 min at 30 °C. The sample (200 μl) was then loaded onto the gel filtration column and separated at 0.2 ml/min at 4 °C. Owing to the low number of aromatic amino acids in Sae2, the protein was monitored by absorbance at 215 nm.
5
Phosphorylation Analysis via Phos-tag SDS-PAGE
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Cells were lysed in immunoprecipitation buffer or in immunoprecipitation buffer lacking phosphatase inhibitors, and lysates were cleared by centrifugation. For λ-phosphatase treatment, supernatants lacking phosphatase inhibitors were adjusted to 1 mM MnCl2, and 800 units of λ-phosphatase (P0753, New England Biolabs) were added for 30 min at 30 °C. Samples were subjected to SDS-PAGE/Western blotting using a 6% acrylamide resolving gel containing 20 μg Phos-tag reagent (Wako Pure Chemical Industries; AAL-107) in the presence of 0.1 mmol/l MnCl2. Following electrophoresis at 60 to 90 V, gels were equilibrated in transfer buffer containing 10 mM EDTA (2 × 10 min). The gels were then soaked in regular transfer buffer for another 10 min followed by immunoblotting.
6
Whole Cell Lysate Dephosphorylation Assay
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In a total volume of 50 µL, 100 µg of whole cell lysate was incubated with 5 µL 10xPMP buffer (New England Biolabs), 5 µL MnCl2, and 800 units λ-phosphatase (NEB) for 1 h at 30 °C.
7
Phosphorylation of WRN Protein by DNA-PKcs
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WRN and Ku proteins were purified to near homogeneity from Sf9 insect cells infected with recombinant baculovirus carrying the respective human cDNAs [24 (link)]. DNA-PKcs was purified from either human placenta or cultured HeLa cells as reported previously [69 (link)]. Purified WRN was dephosphorylated using a mixture of λ-phosphatase and protein phosphatase 1 (New England Biolabs). Purified WRN was phosphorylated in vitro by DNA-PKcs in the presence of Ku70/80 and 50 μM ATP. The phosphorylated WRN band was excised, digested with trypsin, and analyzed by mass spectrometry as described previously [33 (link)].
8
Phosphatase Treatment of Cell Lysates
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Cell lysates were treated with 2 units of λ phosphatase (New England BioLabs) at 30°C for 30 min. SDS sample buffer was added, and the samples were incubated at 98°C for 5 min.
9
Phosphatase-mediated Protein Dephosphorylation
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A total of 120 μg protein extracts were incubated in phosphatase buffer (New England Biolabs) with or without 1600U of λ phosphatase (New England Biolabs) for 30 min at 30°C. After incubation the reaction was diluted with rehydration buffer up to a volume of 200 μl and processed as previously described for 2D-PAGE. Alternatively, immunoprecipitated proteins were incubated with 800U of λ phosphatase for 1 h in phosphatase buffer at 30°C before resuspension in Laemmli buffer.
10
Dephosphorylation of Strep–SPAT-1 Variants
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Dephosphorylation of immobilized Strep–SPAT-1 WT or mutants on Strep-Tactin Sepharose beads was performed for 2 h in λ phosphatase buffer supplemented with MnCl2 and 400 U λ phosphatase (New England Biolabs, Inc.). Then, λ phosphatase was washed away, and subsequent steps were performed in the presence of phosphatase inhibitors (Figs. 1 D and 2 E ).
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