Combinations of wild type and mutated forms of recombinant His-Flag-CIPK26N and His-Flag-KEGRK were incubated at 30°C for 30 min in 30 μL kinase assay buffer adapted from previously described (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1% Triton X-100, and 10 μCi of [γ-33P]ATP) (Ren et al., 2002 (link)). The reaction was stopped by the addition of SDS-loading buffer and boiling for 5 min. Samples were separated on a 7.5% SDS-PAGE gel and the gel was dried with a gel dryer (Biorad) and phosphorylated protein was detected by autoradiography. Kinase assay was repeated twice.
Gel dryer
Manufactured by Bio-Rad
Sourced in United States
The Gel dryer is a laboratory instrument designed to dry polyacrylamide or agarose gels after electrophoresis. It uses controlled heat and airflow to efficiently remove moisture from the gel, allowing for further analysis or storage.
Automatically generated - may contain errors
Lab products found in correlation
Autoradiography cassette, by Bio-Rad (2 mentions)
Bas 1800 2 phosphoimager, by Fujifilm (2 mentions)
T4 pnk, by New England Biolabs (2 mentions)
Fla 5000 phosphoimager, by Fujifilm (2 mentions)
Typhoon phosphorimager, by GE Healthcare (2 mentions)
T4 polynucleotide kinase, by PerkinElmer (2 mentions)
Imagequant software v5, by GE Healthcare (2 mentions)
35s met and 35s cys protein labeling mix, by PerkinElmer (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Fla 7000, by Fujifilm (2 mentions)
Hyperfilm, by Cytiva (2 mentions)
Sybr gold, by Thermo Fisher Scientific (2 mentions)
Amersham typhoon control software 2, by Cytiva (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx stain free precast gel, by Bio-Rad (2 mentions)
Image lab 5, by Bio-Rad (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Fla 7000 phosphorimager, by Fujifilm (1 mentions)
58 protocols using gel dryer
1
In Vitro Kinase Assay for CIPK26N and KEGRK
2
Detecting Chitinase Activity in E. coli
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Crude protein extracts from IPTG induced and uninduced control E. coli cells was separated by 12% SDS-PAGE containing 0.01% glycol chitin. Sample loading buffer was same as that of SDS-PAGE but without β-mercaptoethanol. After electrophoresis, gel was immersed in a 0.1 M sodium acetate buffer (pH 5.0) containing 1% (v/v) deionized Triton X-100 and incubated for 30 min on shaker. The gel was then transferred to a fresh 0.1 M sodium acetate buffer (pH 5.0), and incubated at 37°C in thermostated chamber overnight for renaturation. After incubation, sodium acetate buffer was removed and activity staining was done with silver stain. Fixation was performed in a liquid solution containing 50% methanol, 12% acetic acid, and 0.0185% formaldehyde, all in v/v. Gels were then incubated on a shaker for 10 min in 40% (v/v) ethanol and for 10 min in 30% (v/v) ethanol, in continuous order. Pre-treatment, rinsing, and silver impregnation were performed using standard methods. Then the reaction was stopped using 5% acetic acid. After the development was stopped, gel was washed initially with 30% (v/v) methanol for 20 min and later with 10% (v/v) methanol for 20 min and then stored in 10% (v/v) methanol at 4°C before drying. Silver-stained gel was dried with a gel dryer (Bio-Rad Laboratories, Richmond, CA) and preserved at room temperature (Marek et al., 1995 (link)).
3
Selective 2'-Hydroxyl Acylation Analysis
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The selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) was used as previously described (Wilkinson et al. 2006 (link)). Briefly, 40 ODs of exponentially growing cells were lysed and polysomal profiles were generated as described above. Sub-60S, 60S, and 80S fractions from wild-type and mutant strains were collected, sucrose was removed with PD10 Columns (GE Healthcare), re-concentrated with Amicon Ultra Centrifugal Filter Units (Millipore) and treated with 1-methyl-7-nitroisatoic anhydride (1M7) (Prime Organics, Woburn, US) or DMSO. RNA was extracted using a standard phenol:chloroform extraction. Primer extension was performed with a 32P 5′-end labeled primer (5′-AAAACTAGTGATTCTGCCAAGCCCG-3′) complementary to 25S rRNA 95 nucleotides downstream from C2278. Samples were resolved on a 6% urea-polyacrylamide gel at 20W for 2.5 h. The gel was dried for 1 h at 80°C using a gel dryer (BioRad), exposed to imaging plate (Fujifilm), and scanned on the Fujifilm FLA7000 PhosphorImager.
4
Kinase Activity Assay for EtROP1
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An in vitro kinase activity assay was performed to assess the phosphorylation activity of EtROP1. Kinase assay reactions were performed using 1 μg of purified EtROP1 (WT, mutated or truncated). Recombinant proteins were incubated with heterologous substrates (casein or Myelin Basic Protein, MBP) or crude cell lysate at 37°C for 30 min in a kinase buffer (60‐mM Tris–HCl, pH 7.5; 60‐mM MgCl2; 6‐mM MnCl2, NaF 0.1 M, glycerophosphate 0.3 M) supplemented with 5‐μM unlabelled ATP and 10‐μCi [γ‐32P]ATP (Perkin‐Elmer). After incubation, the reaction was stopped by addition of Laemmli buffer and heat denaturation at 95°C for 5 min. Phosphorylated proteins were separated on 10% SDS‐PAGE gels. The gels were stained with Coomassie Brilliant Blue and placed in a gel dryer (Bio‐Rad), exposed to an X‐ray film in an autoradiography cassette (Bio‐Rad), and visualised by autoradiography.
5
Quinoxaline Binding Assay with FPN
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Induced and uninduced cells were treated ± 3H-quinoxaline at 0.33 μM, and incubated at 37°C for 30 min. Unbound quinoxaline was removed by centrifugation. Non-reduced clarified lysates were run on a 4–12% Bis-Tris gel. The gel was cut into 2 parts; one side was dried on a BioRad gel dryer and exposed to autoradiography film for 1 month. The other side was transferred to nitrocellulose membrane and FPN detected with antibody 38C8.
6
Phosphorylation Assay for Recombinant PKC
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GST-tagged PC1 expression constructs were expressed and purified with glutathione beads as described above. Beads were washed once with lysis buffer, twice with wash buffer (25 mM Tris⋅HCl at pH 7.4, 0.05% Triton X-100, 1 mM CaCl2, 20 mM MgCl2, 1 mM dithiothreitol [DTT]), and then resuspended in 50 μL of kinase reaction buffer per reaction (25 mM Tris⋅HCl at pH 7.4, 0.05% Triton X-100, 1 mM CaCl2, 20 mM MgCl2, 1 mM DTT, 100 μM adenosine triphosphate [ATP], 0.2 mg/mL phosphatidyl serine, 200 nM phorbol-12-myristate-13-acetate, 300 μCi/mL ATP). Beads were incubated for 45 min at 30 °C with either 20 ng of recombinant PKCδ (Calbiochem) or 40 ng PKCζ (Sigma). The reactions were stopped by washing the beads once with ice-cold wash buffer. Phosphorylated proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The gel was dried for 6 h using a gel dryer (Bio-Rad), and radioactive phosphate was detected using a phosphorimager (Bio-Rad). Protein loading of each reaction was determined in parallel by Western blot analysis. All experiments are representative of three independent experiments.
7
Gelatin Zymography for MMP-2 and MMP-9
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Nasal fibroblasts were exposed to CSE for 72 h after pretreatment with ROS scavengers, PI3K/Akt inhibitor and NF-κB inhibitor for 1 h. Aliquots of fibroblast-conditioned medium (10 µL) were analyzed using gelatin zymography for MMP-2 and MMP-9 in 1 mg/mL gelatin-10% polyacrylamide gels. Following electrophoresis, the gels were washed twice with 2.5% Triton X-100 for 30 min while shaking to remove sodium dodecyl sulfate and renature MMP-2 and MMP-9 in the gels. Renatured gels were incubated in developing buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35 overnight at 37 °C. Gels were stained with 0.25% Coomassie brilliant blue G-250 (50% methanol, 10% acetic acid) and destained using destaining solution (50% methanol, 10% acetic acid). Proteinase activity was observed as cleared (unstained) regions on the gels. Finally, the gels were dried for 2 h using a gel dryer (Bio-Rad, Hercules, CA, USA).
8
Kinase Assays with MARP Inhibitors
9
Quantifying Radiolabeled DNA Oligonucleotides
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Radiolabeled DNA oligonucleotides were denatured (95°C in 50% formamide for 3 min) and resolved on a denaturing polyacrylamide gel (15% acrylamide:bis-acrylamide 29:1, 7 M urea, 0.5X TBE). Gels were dried (4 hr, 80°C) on a gel dryer (Bio-Rad) and exposed to a phosphor screen. Phosphor screens were imaged on an Amersham Typhoon phosphorimager (GE Healthcare). Phosphorimages were quantified using ImageQuant software (GE Healthcare).
10
Parkin-PINK1 Kinase Assay Protocol
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Reactions were set up in a volume of 25 μl, using 2 μg of wild-type or indicated mutants of Parkin and 1 μg of E. coli-expressed wild-type or kinase-inactive (D359A) MBP-TcPINK1, in 50 mM Tris–HCl (pH 7.5), 0.1 mM EGTA, 10 mM MgCl2, 2 mM DTT and 0.1 mM [γ-32P] ATP (approx. 500 cpm pmol−1). Assays were incubated at 30°C with shaking at 1050 r.p.m. and terminated after 60 min by addition of SDS sample loading buffer. The reaction mixtures were then resolved by SDS–PAGE. Proteins were detected by Coomassie staining, and gels were imaged using an Epson scanner and dried completely using a gel dryer (Bio-Rad). Incorporation of [γ-32P] ATP into substrates was analysed by autoradiography using Amersham hyperfilm.
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