Gemin5 protein or its tagged-domains were immunodetected using anti-Gemin5 (Novus) antibody. P0 was detected with the monoclonal antibody 3BH5 (46 (link)). Commercial antibodies were used to detect eIF3b, eIF4B, DHX9 (Bethyl laboratories), His-tag, tubulin (Sigma), PTB (Acris), hnRNPA1, hnRNPU (Immuquest), CBP (Abcam), GST, RACK1, RPL3, RPL4 (Santa Cruz), eIF4E (Transduction laboratories). Appropriate secondary antibodies (Thermo Scientific) were used according to the manufacturer instructions. Protein signals were visualized with ECL plus (Millipore). Quantification of the signal detected was done in the linear range of the antibodies.
His tag
Manufactured by Merck Group
Sourced in United States
His-tag is a commonly used peptide tag that can be fused to the N- or C-terminus of a recombinant protein. The His-tag consists of a series of histidine residues, typically 6 or 10, that facilitate the purification of the tagged protein using immobilized metal affinity chromatography (IMAC).
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase labeled secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Cxcr4 and α sma, by Abcam (2 mentions)
Tenascin c, by Santa Cruz Biotechnology (2 mentions)
Subcellular protein fractionation kit, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti gemin5, by Novus Biologicals (1 mentions)
Ecl plus, by Merck Group (1 mentions)
Appropriate secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rack1, by Santa Cruz Biotechnology (1 mentions)
Ptm 502, by PTM Biolabs (1 mentions)
Crotonyl coa, by Merck Group (1 mentions)
Bacterial adenylate cyclase two hybrid system kit, by Euromedex (1 mentions)
Gst tag, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
In fusion cloning, by Takara Bio (1 mentions)
Pacbio rs 2 system, by Pacific Biosciences (1 mentions)
Kod polymerase, by Merck Group (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
8 protocols using his tag
1
Immunodetection of Gemin5 and Associated Proteins
2
Bacterial Two-Hybrid Crotonylation Assay
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Bacterial two hybrids for protein–protein interaction were performed with the Bacterial Adenylate Cyclase Two-Hybrid System Kit (Euromedex) according to the product manual. For in vitro crotonylation reaction, 1 µg of Kct1, 2 µg of Glk, and 10 mM of crotonyl-CoA (Sigma) were added in the reaction buffer (20 mM Tris-HCI (pH 8.0), 100 mM KCl, 7.5 mM MgCl2) and incubated at 30 °C for 1.5 h, followed by Glk activity assays or loading for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Decrotonylation assays were performed in the same reaction conditions, but 2.5 µg of Glk + 2 µg of CobB were used. The total loading protein and crotonylation were analyzed by western blot with antibodies against His-tag (Sigma) and crotonyllysine (Kcr) (PTM-502, PTM Biolabs Inc.) (1:2000), respectively.
3
Protein Expression Analysis Workflow
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Whole cell lysates were prepared by scraping cells directly in 2× sodium dodecyl sulfate (SDS) sample buffer. In parallel experiments, membrane proteins were extracted using the Subcellular Protein Fractionation kit (Pierce Biotechnology, Rockford, IL) following the manufacturer's instructions. Extracellular matrix was prepared as we previously described.4 Proteins were separated by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to PVDF membranes. Membranes were blocked with 5% nonfat dry milk in Tris‐buffered saline (TBS)/0.05% Tween‐20 (TBST) buffer then incubated with antibodies against human IGFBP‐4, collagen, fibronectin, tenascin‐C, CTGF, GAPDH (Santa Cruz, Dallas, TX), phosphorylated SMAD2 and SMAD3, total SMAD2 and SMAD3, phosphorylated SMAD1/5/9, phosphorylated (p)‐44/42 MAPK, p‐AKT, p‐P38 kinase, p‐SAPK/JUNK, and antibodies targeting the corresponding total proteins (Cell Signaling Tech, Danvers, MA), CXCR4 and α‐SMA (Abcam, Cambridge, MA), His tag (Sigma), or tubulin (Epitomics Inc, Burlingame, CA), washed with TBS three times, and then incubated with horseradish peroxidase‐labeled secondary antibody (Santa Cruz). Signals were detected by chemiluminescence (Perkin Elmer, Waltham, MA, USA) on a FluorChem R system (ProteinSimple, San Jose, CA). Images were analyzed using Image J.
4
Western Blot Analysis of Bait and Prey Proteins
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Antibodies were to the His-tag (Sigma), GFP (Clontech) or commercially produced polyclonal antibodies (Invitrogen) raised in rabbits to the following specific oligopeptides: PbGpb1—CDIRADRELNTYQSD or PbTpk2 –SQFDRYPEETEPYG were used. Proteins were separated by SDS-PAGE (on 4 to 12% polyacrylamide gels) and electrotransferred to PVDF membrane. Blots were incubated with the respective antibodies (e.g anti-His6 at 1:5000 dilution; anti-GFP at 1:100 dilution and PbGpb1 and PbTpk2 specific antibodies at 1:2500 dilution). Alkaline-phosphatase-conjugated goat anti-mouse IgG (1:2500 dilution) and horse radish peroxidase-conjugated anti-rabbit IgG (1:2500 dilution) were used as secondary antibodies for His6, GFP, mRFP and specific-protein blots, respectively. Western-blots were used routinely to confirm the presence of bait and prey proteins.
5
Arabidopsis Protein Extraction for Immunoblot
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Four‐week‐old Arabidopsis leaf tissue (20–30 mg) was frozen in liquid nitrogen and homogenized. The samples were dissolved in 200–300 μl 2 × SDS–PAGE sample buffer (100-mM Tris/HCl pH 6.8, 4% SDS, 20% glycerol, and 2-mM dithiothreitol) at 95°C for 10 min and centrifuged for 1 min at 13,053 rpm at room temperature. Aliquots (10–15 μL) of each sample were then loaded onto an SDS–PAGE for subsequent immunoblot analysis. The antibodies were either purchased (Thermo Fischer [GST-tag] and Sigma [His-tag]) or produced from purified antigens (GSAAT, GluTR, GBP, FLU, ALAD, coproporphyrinogen oxidase [CPOX], PPOX, FC2, FC1, genomes uncoupled 4 [GUN4], and PORB) generated in our laboratory.
6
Protein Extraction and Analysis Workflow
7
Codon-Optimized Protein Expression in E. coli
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Amino acid sequences were
codon optimized for expression in E. coli using an
empirically derived codon usage table.29 (link) Optimized DNA, gene partitioning, and construction oligos were generated
by GeneDesign.30 (link) Oligonucleotides were
pooled for synthesis using a Hamilton STAR liquid handler (Hamilton
Robotics). Assembly proceeded in a two-step PCA reaction in 25 μL
final volume using KOD polymerase (EMD Millipore). Assembled products
were cloned into pET45b(+) vector containing an N-terminal His-Tag
(EMD Millipore) by In-Fusion cloning (Clontech) and transformed into
BL21 cells in 96-well plates. Plating and picking was performed using
a QPix 400 system (Molecular Devices). Eight colonies per construct
were sequenced verified using PACBIO RSII system (Pacific Biosciences).
codon optimized for expression in E. coli using an
empirically derived codon usage table.29 (link) Optimized DNA, gene partitioning, and construction oligos were generated
by GeneDesign.30 (link) Oligonucleotides were
pooled for synthesis using a Hamilton STAR liquid handler (Hamilton
Robotics). Assembly proceeded in a two-step PCA reaction in 25 μL
final volume using KOD polymerase (EMD Millipore). Assembled products
were cloned into pET45b(+) vector containing an N-terminal His-Tag
(EMD Millipore) by In-Fusion cloning (Clontech) and transformed into
BL21 cells in 96-well plates. Plating and picking was performed using
a QPix 400 system (Molecular Devices). Eight colonies per construct
were sequenced verified using PACBIO RSII system (Pacific Biosciences).
8
Immunoblotting of Viral Antigen Proteins
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Antigen preparations were resolved by SDS-PAGE, transferred to PVDF membranes (EMD Millipore), and probed with antibodies as published [28] (link). In some experiments, antigens resolved by SDS-PAGE were stained with SimplyBlue™ SafeStain (ThermoFisher Scientific). The mouse antibody His-Tag (EMD Millipore) was used in experiments with His-Tag BatA protein. Murine antibodies against a peptide encompassing BatA residues 208–221 (GenScript custom polyclonal antibody production services) were used in experiments with PIV5-BatA virus. Goat anti-mouse antibodies conjugated to HRP (SouthernBiotech) were utilized for detection.
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