CPR crosslinking assays (Figure 5 ) were performed as described previously (Nayak et al., 2013 (link)). Nucleic acid scaffolds were prepared by annealing RNA, template DNA (T-DNA) and 15 μM non-template DNA (NT-DNA) at 10 µM, 12 µM, and 15 µM final concentrations, respectively, in reconstitution buffer (RB; 20 mM Tris-HCl pH 8, 20 mM NaCl, and 1 mM EDTA). ECs, ePECs, and his PEC were formed by incubating 1 μM RNAP and scaffold (2 μM, based on RNA) in buffer A (50 mM Tris-HCl pH 8, 20 mM NaCl, 10 mM MgCl2, 1 mM EDTA, and 2.5 ug acetylated bovine serum albumin/mL for 15 min at room temperature (RT). For crosslinking reactions with NTP, 3′deoxyECs formed by reaction with 3′dNTP were incubated for 15 min at RT with 0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 mM GTP or ATP. Next, EC, ePEC, or his-PEC (final RNAP 0.8 uM and scaffold 1.6 uM) were incubated for 60 min with 2.5 mM CSSC and 0.05 mM DTT (E = –0.16) and stopped with 50 mM iodoacetamide. Samples were separated by native PAGE to verify reconstitution efficiency and by sodium dodecyl sulfate (SDS)-PAGE using 4–15% GE Healthcare PhastGel to quantify formation of crosslinks. Gels were stained with Coomassie Blue and imaged with a CCD camera. The fraction cross-linked was quantified with ImageJ software.
Phastgel
Manufactured by GE Healthcare
Sourced in Sweden, United States
Phastgel is a laboratory equipment product from GE Healthcare. It is designed for the separation and analysis of proteins using electrophoresis technology. The core function of Phastgel is to provide a reliable and efficient platform for protein separation and characterization.
Automatically generated - may contain errors
Lab products found in correlation
14 protocols using phastgel
1
RNAP Crosslinking Assay Protocol
2
Native Gel Electrophoresis of Core-Antibody Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were mixed to achieve a final concentration of 10 µM coreS (as trimer, unless stated otherwise) and appropriate molar ratio of antibody as indicated. This concentration was chosen because it represents a good detection limit to differentiate between the binding of neutralizing and non-neutralizing Fabs and ScFvs. They were incubated at room temperature for 30 min and 2 µl of sample was layered for electrophoresis on a 20% homogeneous Phastgel using 1 µl/eight-lane applicators (unless stated otherwise) and native buffer strips (GE Healthcare). Gels were stained in Phastgel Blue R, destained and digitized. For detection at lower concentrations, 4 µl/six-lane applicators and PlusOne silver staining kit (GE Healthcare) were used.
3
CPR Crosslinking Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CPR crosslinking assays (Figures 3 , 6 and 7 ) were performed as described previously (17 (link)). Nucleic acid scaffolds were prepared by annealing 10 μM RNA, 12 μM template DNA (T-DNA) and 15 μM non-template DNA (NT-DNA) in reconstitution buffer (20 mM Tris-HCl pH 8, 20 mM NaCl and 1 mM EDTA). ECs (elongation complexes) and paused elongation complexes (PECs) were formed by incubating 1 μM RNAP and 2 μM scaffold in buffer A (50 mM Tris-HCl pH 8, 20 mM NaCl, 10 mM MgCl2, 1 mM EDTA and 2.5 ug/ml acetylated bovine serum albumin (BSA)) for 15 min at room temperature (RT). For crosslinking reactions with NTP, ECs were incubated for 15 min at RT with 10 mM GTP. Free RNAP, EC, or PEC (final RNAP 0.8 uM and scaffold 1.6 uM) were incubated for 60 min with 2.5 mM CSSC and 0.05–20 mM DTT (E = –0.140 to –0.414 V; 0.1 mM CSSC and 0.002–0.8 mM DTT was used for U937–1139 RNAP) and stopped with 50 mM iodoacetamide. Samples were separated by native PAGE to verify reconstitution efficiency and by sodium dodecyl sulphate-PAGE (SDS-PAGE) 7.5% GE Healthcare PhastGel (10–15% PhastGel for any U937–1139 RNAP) to quantify formation of crosslinks. Gels were silverstained or Coomassie brilliant blue R-250 stained, image digitized (FluorChem CCD; Protein Simple, Inc.) and quantified (ImageJ).
4
Characterization of hVDR-hWBP4 Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant hVDR full-length was mixed with a 5-fold molar excess of hWBP4 polypeptide (amino acid 122-196) in the presence of two equivalents of ZK. Then, 5 μg of proteins were loaded on a 4–15% gradient GE Healthcare PhastGel™ with Native PhastGel Buffer Strips (GE) following the manufacturer’s protocol and revealed by Coomassie Brilliant Blue (CBB) staining.
5
Transferrin Iron Saturation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IEF of serum or plasma Tf was performed essentially as described by Van Eijk and Van Noort.13 In short, 10 μL plasma was incubated with an Fe(III)-citrate solution to saturate Tf with Fe(III). Supernatant (1 μL) was subsequently applied on a polyacrylamide gel with a pH gradient of 4.0–6.5 (PhastGel, GE Healthcare Bio-Sciences). After electrophoresis (2000 V), Tf is immunofixated using Rabbit antihuman Tf and coloured using Coomassie Brilliant Blue R250.
6
Cysteine-Pair Cross-Linking Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Cys triplet reporter (CTR) cross-linking assays (Figure 6B,E and F ), ECs and PECs were assembled on purified DNA and RNA scaffolds specified in the figure legend and as described previously (Kang et al., 2018a (link)). Briefly, 10 μM RNA, 12 μM template DNA, and 15 μM non-template DNA (Supplementary file 1 ) were annealed in RB. To assemble complexes, scaffold (2 μM) was mixed with limiting CTR RNAP (1 μM; CTR RNAP: β′1045iC 258iC, β843C) in 50 mM Tris-HCl, pH 7.9, 20 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, 5% glycerol, and 2.5 μg of acetylated bovine serum albumin/mL, and added to mixtures of cystamine and DTT to generate redox potentials that ranged from −0.314 to −0.424. Complexes were incubated for 60 min at room temperature and then were quenched with the addition of iodoacetamide to 15 mM. The formation of cysteine-pair cross-links was then evaluated by non-reducing SDS-PAGE (4%–15% gradient Phastgel; GE Healthcare) as described previously (Nayak et al., 2013 (link)). Gels were stained with Coomassie Blue and imaged with a CCD camera. The fraction cross-linked was quantified with ImageJ software. The experimental error was determined as the standard deviation of measurements from three or more independent replicates.
7
Purification and Characterization of Hb from S. microcephalus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hb purification was achieved by anion-exchange chromatography at 4°C on a Mono Q-Tricorn column (1.0 ×10 cm) mounted on an AKTA-FPLC system (Pharmacia, Uppsala, Sweden). The column was equilibrated with 20 mM Tris-HCl, pH 8.0 (buffer A); Hb elution was performed with a gradient from buffer A to buffer B (80 mM NaCl, pH 8.0, in buffer A). The major Hbs, namely Hb 1, Hb 2 and Hb 3, were eluted at 25, 35 and 50% buffer B, respectively. To improve the separation, the hemolysate was treated with 10 mM dithiotreitol (DTT) overnight at 4°C under anaerobic conditions. Hb multiplicity and purity were analysed by isoelectrofocusing (IEF, pH 3.0 to 9.0) on polyacrylamide gels (Phastgel, GE Healthcare Biosciences AB, Uppsala, Sweden). Hbs were saturated with CO and stored at −20°C.
The S. microcephalus Hb concentration was estimated spectrophotometrically at 540 and 569 nm, using the millimolar extinction coefficient of the ferrous carbonylated-heme (ε = 13.4 mM−1cm−1) [25 ].
The S. microcephalus Hb concentration was estimated spectrophotometrically at 540 and 569 nm, using the millimolar extinction coefficient of the ferrous carbonylated-heme (ε = 13.4 mM−1cm−1) [25 ].
8
Desialylation and Leg5Ac7Ac Modification of α1-Antitrypsin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The glycoprotein (6 mg) was desialylated in MES buffer pH 6.0 (1 mL of 0.5 M) with 25 mU of M. viridifaciens sialidase, at 37 o C for 4 h. Removal of the sialic acid residues was confirmed by iso-electric focussing (IEF), carried out with a Pharmacia (GE Healthcare) Phastgel apparatus, using precast Phastgel pH 3-9, and manual staining with Coomassie Blue. The product was diluted to 50 mL with 20 mM Tris buffer pH 7.5 and purified by ion-exchange chromatography on a 5ml HiTrap Q HP column, run in the same Tris buffer and eluted with a 0-0.3M NaCl gradient.
The asialo-α1-antitrypsin was modified with Leg5Ac7Ac by incubation of 0.5 mL of the protein (3.03 mg/mL) with 100 µl of 0.5 M MES buffer, pH 6.5, 100 µL of 100 mM MgCl2, 300 µL of 10 mM CMP-Leg5Ac7Ac and 100 µL of ST6Gal1, for 20 h at 30 o C; a second 300 µL aliquot of the CMP-Leg5Ac7Ac was added after 4 h. The reactions were checked by IEF. The reaction mixture was diluted with 100 mL of 20 mM Tris buffer pH 7.5, and the product was purified by ion exchange as described above. The removal of sialic acid residues and the addition of Leg5Ac7Ac were quantified by mass spectrometry.
The asialo-α1-antitrypsin was modified with Leg5Ac7Ac by incubation of 0.5 mL of the protein (3.03 mg/mL) with 100 µl of 0.5 M MES buffer, pH 6.5, 100 µL of 100 mM MgCl2, 300 µL of 10 mM CMP-Leg5Ac7Ac and 100 µL of ST6Gal1, for 20 h at 30 o C; a second 300 µL aliquot of the CMP-Leg5Ac7Ac was added after 4 h. The reactions were checked by IEF. The reaction mixture was diluted with 100 mL of 20 mM Tris buffer pH 7.5, and the product was purified by ion exchange as described above. The removal of sialic acid residues and the addition of Leg5Ac7Ac were quantified by mass spectrometry.
9
In vitro Kv7.2 and A5C Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GST‐Kv7.2C and GST‐A5C were phosphorylated in vitro with a mixture of recombinant CDK5, p38 MAPK, PKA and CaMKIIα kinases in the presence or absence of four protein kinase inhibitors (1 μm roscovitine, 3 μm SB203580, 10 μm KN‐62 and 10 μm H7; KI‐MIX). Each reaction was performed with 7.5 μg GST‐fusion protein in kinase buffer (50 mm Tris pH 7.6, 6.25 mm DTT, 47 mm MgCl2, 33 mm CaCl2, 8 mm CaM, 625 μm cAMP, 93.5 ng CaMKIIα, 100 ng CDK5, 144 ng p38 MAPK and 101.75 ng PKA) with solvent or KI‐MIX. A mixture of hot ([γ‐32P]ATP, Hartmann Analytics, Braunschweig, Germany) and cold ATP was added at a final ratio of 1:3, resulting in 3.3 pmol of total ATP in each reaction. All steps were carried out on ice until ATP was added, and the tubes were incubated at 30°C for 30 min. Subsequently, the reactions were filled with 50 mm Tris buffer (pH 7.6) and 3 μg of anti‐Kv7.2 antibody (NeuroMab) added for immunoprecipitation. Extracts were used to count the radioactivity in a Packard 1900CA Tri‐Carb liquid scintillation analyser (PerkinElmer, Waltham, MA, USA) and for autoradiography on a 12.5% PhastGel (GE Healthcare).
10
In vitro kinase phosphorylation of Kv7.2 and A5C
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!