ATPase assays were performed as described previously using condition B described by Lorsch and Herschlag (Lorsch and Herschlag, 1998 ). Briefly, 1.5 μM eIF4A1 was incubated with indicated compound, 5 μM poly (AG)8 RNA, 500 μM cold ATP in the presence of buffer containing 2.5 mM MgCl2, 1mM DTT, 1% glycerol, 20 mM MES-KOH [pH 6.0], 10 mM KOAc for 30 minutes before adding 1 μCi [γ32P]-ATP (3000Ci/mmol). At each time point (0, 30, 60 90, 120 mins), 2 μl of the reaction was put on ice and quenched with a final concentration of 10 mM EDTA. The reactions were then resolved on PEI cellulose TLC plates (Millipore) developed with 1M LiCl, 0.3M NaH2PO4. The extent of ATP hydrolysis was quantitated using a Storm 840 (Molecular dynamics) scanner.
Storm 840
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Israel
The Storm 840 is a laboratory equipment product from GE Healthcare. It is designed for the separation and purification of biomolecules, such as proteins and nucleic acids, using chromatography techniques. The product's core function is to facilitate the isolation and concentration of the desired biomolecules from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Amicon ultra 100k, by Merck Group (2 mentions)
Anti flag m2 affinity resin, by Merck Group (2 mentions)
Imagequant 5, by GE Healthcare (2 mentions)
Cfsa 1x, by Leica (2 mentions)
Phosphor imaging screen plate, by GE Healthcare (2 mentions)
Novex sharp prestained, by Thermo Fisher Scientific (2 mentions)
Pei cellulose tlc plate, by Merck Group (1 mentions)
Carbo free blocking solution, by Vector Laboratories (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Molecular mass standards, by Thermo Fisher Scientific (1 mentions)
Wet transfer unit, by Hoefer (1 mentions)
Storage phosphor screen, by GE Healthcare (1 mentions)
Phosphostorage screen, by GE Healthcare (1 mentions)
γ 32p atp, by Hartmann Analytic (1 mentions)
Anti v5, by Thermo Fisher Scientific (1 mentions)
Anti pcna, by Merck Group (1 mentions)
Secondary antibodies coupled to alkaline phosphatase, by Jackson ImmunoResearch (1 mentions)
Nanoject 2, by Drummond (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Fla 9000, by Fujifilm (1 mentions)
23 protocols using storm 840
1
ATPase Activity Assay of eIF4A1
2
RNA Analysis of FGFRL1 and GAPDH
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Total RNA was isolated from the confluent cell cultures with the GenElute miniprep kit from Sigma-Aldrich (St. Louis, MO, USA). The RNA was resolved on 1% agarose gels in the presence of formaldehyde. The gels were stained with ethidium bromide to visualize the 18S and 28S ribosomal RNAs. The RNAs were quantitatively transferred to a nylon membrane by vacuum blotting. The blot was hybridized under standard conditions with probes that contained full-length cDNAs for FGFRL1 and GAPDH, respectively. Beforehand, these probes had been radiolabeled by random primed oligolabeling with 32P-dCTP (New England Nuclear/PerkinElmer, Waltham, MA, USA). The blot was extensively washed with 1X SSC and exposed to X-ray film (Carestream Kodak BioMax MS; Sigma). For quantification of the bands, the blot was scanned with a phosphorimager (Storm 840; Molecular Dynamics, Sunnyvale, CA, USA).
3
Purification and Immunoblotting of RubisCO
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The R. rubrum RubisCO, MTA phosphorylase, MTR-1P isomerase, and MTRu-1P isomerase (RLP) were purified as described previously (12 (link)). For immunoblotting analysis of RubisCO, purified protein and crude extracts were assayed as previously described (28 (link)). Immunoblots were developed with the Attophos (Amersham, Buckinghamshire, England) detection reagent according to the manufacturer’s instructions and visualized with a Molecular Dynamics Storm 840 imaging system.
4
Salivary Glycoprotein Profiling by SDS-PAGE and Lectin Blotting
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The pooled salivary proteins from HV, BB, BC-I, and BC-II subjects of the retrospective cohort were analyzed by SDS-PAGE and subsequently lectin blotting as previously described (Qin et al., 2013 (link), Zhong et al., 2015 (link)). Briefly, For SDS-PAGE, samples were boiled for 4 min at 100 °C mixed with 5 × loading buffer, and run on a 10% polyacrylamide resolving gel and a 3% stacking gel. Molecular mass standards (Thermo Scientific, Waltham, USA) were run with all gels. Some gels were then stained directly with alkaline silver. For lectin blotting, the proteins in gels were then transferred to a PVDF membrane (Immobilon-P; Millipore Corp., Bedford, MA, U.S.A.) with a wet transfer unit (Hoefer Scientific) for 1.5 h at 32 mA. After transfer, the membranes were washed twice with TTBS (150 mM NaCl, 10 mM Tris-HCl, 0.05% v/v Tween20, pH 7.5) and then blocked for 1 h with Carbo-Free Blocking Solution (Vector, Burlingame, CA) at room temperature. The membranes were then washed again and incubated with Cy5 (GE Healthcare, Buckinghamshire, UK) labeled lectins (2 μg/mL in Carbo-Free Blocking Solution) with gentle shaking overnight at 4 °C in the dark. The membranes were then washed twice each for 10 min with TTBS and scanned by red fluorescence channel (635 nm excitation/650LP emission) with the voltage of 800 PMT using a phosphor imager (Storm 840, Molecular Dynamics).
5
Quantitative Analysis of AP Site Repair
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This assay was performed as previously described using a 42-mer substrate U21•G containing a single AP site37 (link). Total cell extracts were incubated with the substrate and the reaction products were separated on 10% (w/v) polyacrylamide/7M urea gels and developed by autoradiography. The radiolabeled fragments were visualized by using a PhosphorImager (Storm 840, Molecular Dynamics), and ImageQuant LAS 400 was used for quantification. The intensity of the product was quantified using ImageJ and plotted as a percentage of increasing concentration of the total cell extracts.
6
Cellular Uptake of [18F]pFBG and [18F]FBBG
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A total of 1 × 106 cells were incubated with 3 MBq of [18F]pFBG or [18F]FBBG in 250 µL medium for 15 min at 37 °C (or medium only for control). After washing with 1 mL of complete medium, 200 µL RIPA buffer with protease inhibitor were added and the samples were incubated for 10 min at room temperature. A total of 20 µL of sample were mixed with 4 µL 6× reducing loading buffer, heated to 95 °C for 5 min and SDS-PAGE was performed as described above. A storage phosphor screen (Molecular Dynamics, Caesarea, Israel) was exposed to the gel in a light-shielded cassette for approximately 10 half-lives of 18F (18 h), and the screen was scanned using a phosphor imager (Storm 840, Molecular Dynamics). As a loading control, a part of the gel was stained with a commercial Coomassie solution (InstantBlue Protein Stain, Expedeon/Biozol, Eching, Germany) according to the manufacturer’s instruction.
7
Wdr62 Protein Purification and Kinase Assay
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Flag-Wdr62 plasmids were transfected into 293T cells and selected by puromycin (5 ug/ml) to generate stable cell lines. Crude lysate was collected in the lysis buffer (20 mM Tris-HCl [pH 7.5], 137 mM NaCl, 1mM EDTA, 1.5 mM MgCl2, 10% Glycerol, 0.2% Triton X-100). To purify the Flag-Wdr62 proteins, cytoplasmic extract was incubated with anti-Flag M2 affinity resin (Sigma). After extensive washing with wash buffer (20 mM Tris-HCl [pH 7.5], 0.8 M NaCl), the affinity column was eluted with 400 ug/ml Flag peptide. Proteins were further separated with Superose 6 gel filtration chromatography. Fractions were examined by SDS-PAGE analysis, and fractions containing Wdr62 protein were combined and concentrated with Millipore Amicon Ultra (100K). Purified proteins were snap-frozen in liquid nitrogen and stored at -80°C.
For in vitro kinase assay, purified Flag-Wdr62 proteins or commercially available Tpx2 proteins (SignalChem, cat# T40-30H-20) and Aurora A proteins (SignalChem, cat# A28-18G) were incubated with 25 uM ATP and 4 uCi γ-[32P]-ATP in 20 ul of reaction mixture containing 50 mM Tris-HCl, pH 7.5, 25 mM NaCl, 1 mM DTT, and 10 mM MgCl2. After reacting for 15 min at 30°C, the protein-loading buffer was added and boiled for 5 min for SDS-PAGE gel separation. Gels were autoradiographied with Storm 840 (Molecular Dynamics).
For in vitro kinase assay, purified Flag-Wdr62 proteins or commercially available Tpx2 proteins (SignalChem, cat# T40-30H-20) and Aurora A proteins (SignalChem, cat# A28-18G) were incubated with 25 uM ATP and 4 uCi γ-[32P]-ATP in 20 ul of reaction mixture containing 50 mM Tris-HCl, pH 7.5, 25 mM NaCl, 1 mM DTT, and 10 mM MgCl2. After reacting for 15 min at 30°C, the protein-loading buffer was added and boiled for 5 min for SDS-PAGE gel separation. Gels were autoradiographied with Storm 840 (Molecular Dynamics).
8
Brain Sections Autoradiography with 64Cu-labeled BFC
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Brain sections of 15
month old Tg2576 transgenic mice and aged-matched WT mice were obtained
as described previously48 (link),49 (link) and immersed into a
cryoprotectant solution. These sections were sorted and carefully
removed using phosphate buffer in saline (PBS) with 1% tween-20 solution
and mounted onto adhesive glass slides (CFSA 1X, Leica Bio Systems).
Each section was washed with 100% PBS three times, and ∼0.925
MBq (25 μCi) of 64Cu-labeled BFC in 100 μL
total volume was added to completely cover the brain section and incubate
for 1 h at room temperature in a shielded bunker. After the incubation,
brain sections were washed using PBS with five 1 min cycles and briefly
air-dried. The imaging slides were mounted onto a phosphor imaging
screen plate (GE Healthcare Life Sciences) and exposed for 1–5
min. The plates were scanned using a phosphor imager plate scanner
(Storm 840), and the resulting images were processed using ImageQuant
5.2 (Molecular Dynamics) and ImageJ (v1.48, public domain) software.
month old Tg2576 transgenic mice and aged-matched WT mice were obtained
as described previously48 (link),49 (link) and immersed into a
cryoprotectant solution. These sections were sorted and carefully
removed using phosphate buffer in saline (PBS) with 1% tween-20 solution
and mounted onto adhesive glass slides (CFSA 1X, Leica Bio Systems).
Each section was washed with 100% PBS three times, and ∼0.925
MBq (25 μCi) of 64Cu-labeled BFC in 100 μL
total volume was added to completely cover the brain section and incubate
for 1 h at room temperature in a shielded bunker. After the incubation,
brain sections were washed using PBS with five 1 min cycles and briefly
air-dried. The imaging slides were mounted onto a phosphor imaging
screen plate (GE Healthcare Life Sciences) and exposed for 1–5
min. The plates were scanned using a phosphor imager plate scanner
(Storm 840), and the resulting images were processed using ImageQuant
5.2 (Molecular Dynamics) and ImageJ (v1.48, public domain) software.
9
Radioactive Phosphorylation Assay of ArnB
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In vitro phosphorylation assays using [γ-32P]ATP (Hartmann Analytic) were performed as described (13 (link)). For phosphorylation of purified ArnB and ArnBΔ316, the kinases ArnC and ArnD were used. 2 μm of protein was mixed with 2 μm of ArnC or 0.2 μm of ArnD in a 5× reaction buffer. For assays with ArnD the reaction buffer contained 125 mm MES, pH 6.5, and 750 mm KCl. For assays with ArnC the buffer contained 250 mm HEPES, pH 7.8, and 750 mm KCl. Finally, a mix consisting of 0.8 mm nonradioactive ATP and 0.3 mm [γ-32P]ATP was added to the reaction. After incubation at 55 °C for 10 min, proteins were separated on 11% SDS gels and exposed on a phosphostorage screen (Molecular Dynamics) overnight. Screens were scanned using a phosphorimaging device (Storm 840, Molecular Dynamics).
10
Wdr62 Protein Purification and Kinase Assay
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