Interaction of Hsp90So WT or mutants with DnaKSo was performed by copurification assays with calmodulin beads as previously described (15 (link)). Briefly, E. coli MG1655Δhsp90Ec strains containing plasmids pCBP-hsp90So WT or mutants, and pdnaKSo-6His were grown at 37 °C to an optical density at 600 nm (OD600) of 0.8. As negative controls, plasmids pBad33-CBP and pBad24 were used. Then, 0.05% arabinose was added to allow production of CBP-Hsp90So and DnaKSo-6His for 1 h. Cells were collected, lysed by French press in CBP buffer (150 mM Tris HCl, pH = 8, 150 mM NaCl, 1 mM Mg acetate, 1 mM imidazole, 2 mM CaCl2, 0.1% Triton, 10 mM β-mercaptoethanol), and centrifuged, and the supernatant was incubated for 1 h with 50 µL of CBP affinity resin (Agilent). After four washes with CBP buffer, the resin was resuspended in 40 µL of loading buffer and was heat denatured at 95 °C for 10 min. Purified protein samples and protein extracts before purification (input) were loaded on SDS-PAGE and transferred by Western blot. Hsp90So was detected with an anti-Hsp90Ec antibody (57 (link)) and DnaKSo with an anti-His antibody (Thermo). Copurifications between TilS and Hsp90So were performed as described (15 (link)).
Anti his antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy
The Anti-His antibody is a laboratory tool used to detect and purify proteins that have been modified to include a histidine (His) tag. The antibody binds specifically to the His tag, allowing the target protein to be isolated and identified in various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pip strips, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hispur cobalt column, by Thermo Fisher Scientific (2 mentions)
Mini edta free protease inhibitor tablet, by Roche (2 mentions)
Anti flag antibody, by Thermo Fisher Scientific (2 mentions)
Ubiquitin, by R&D Systems (1 mentions)
Lds loading buffer, by Thermo Fisher Scientific (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Magnetic bead, by Thermo Fisher Scientific (1 mentions)
Gst magnetic beads, by Promega (1 mentions)
Cortactin, by Santa Cruz Biotechnology (1 mentions)
β1 integrin antibody, by BD (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bcecf, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Activated dna, by BPS Biosciences (1 mentions)
Anti gst antibody, by GenScript (1 mentions)
Anti his antibody, by GenScript (1 mentions)
39 protocols using anti his antibody
1
Hsp90-DnaK Interaction Assay
2
Assaying Gli3 Ubiquitination Kinetics
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Gli31–455 ubiquitination was assayed in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, 5 mM ATP, 1 mM DTT, and 2 mg/ml BSA at room temperature at time points from 0 to 30 min. The reaction mixture contained ubiquitinating enzymes at final concentrations of 0.25 μM UBA1 (E1), 8 μM UbcH5B (E2), 2 μM NEDD8‐CUL3‐Rbx1 (E3), 2 μM SPOP28–359 or mutant (substrate adaptor), 75 μM ubiquitin (Boston Biochem, cat. # U‐100H), and 5 μM His6‐tagged Gli31–455. E1, E2, and E3 were purified as described previously (Zhuang et al, 2009 ). The substrate and products were visualized by Western blotting with anti‐His antibody (Thermo Scientific, cat. #MA1‐21315).
3
PNA Inhibition of RickA Protein Expression
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The region starting 100 base pairs upstream of the start codon through 600 base pairs downstream of the rickA gene was amplifed using rickA forward primer 5’- ATTCGTTCATCTATCTTTTTTTTATTTATC-3’ and reverse primer 5’-TTTTTGTATTTCTTTAAGTTCTTTGACATTAG-3’ and cloned into pEXP5-CT (Life Technologies, Grand Island, NY). Translation was performed using the Expressway Cell-Free E. coli Expression System (Life Technologies). PNA targeted to rickA was added to the reaction to demonstrate PNA specificity for its target sequence as measured by impaired protein production. The CALML3 control vector was included in each reaction to serve as a loading control and to demonstrate PNA specificity. The total volume of each sample was resuspended in LDS loading buffer (Life Technologies) with reducing agent, heated for 10 minutes at 70°C and run on NuPAGE 4–12% Bis-Tris gels. Gels were transferred to PVDF membranes using an iBlot gel transfer device (Life Technologies). Membranes were probed using an anti-His antibody (1:3000) (Thermo Fisher Scientific, Waltham, MA) and developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.).
4
Protein-Protein Interaction Assay
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Based on the method reported by Wang et al. (2021a (link), b (link), c (link)), the combination of CmBBX7-GST and CmBBX8-HIS or GST-empty and CmBBX8-HIS was incubated for 2 h at 4 °C after protein induction, and pre-washed GST magnetic beads (Promega, Madison, WI, USA) were added and incubated overnight at 4 °C. The beads were then washed at least three times with wash buffer to remove the heteroproteins, the GST-tagged proteins bound to the magnetic beads were eluted using reduced GST, and the eluted proteins were used for measuring protein–protein interactions by western blotting with anti-His antibody (Thermo Fisher, Waltham, MA, USA), as reported previously (Francisco-Velilla et al. 2016 (link)).
5
Quantification of CD69-S100A8/A9 Binding
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A 10 nm S100A8/S100A9‐tetramer concentration (HBS plus 250 µm CaCl2) was coated to the bottom of a 96‐well plate. After three washing steps, unspecific binding sites were blocked by 0.25% BSA/HBS plus 250 µm CaCl2. Afterward, His‐tagged‐CD69 (R&D) was added at increasing concentrations as indicated in the figure. After 1 h, unbound CD69 was removed by three washing steps and an anti‐His‐antibody (10 µg mL−1, Thermo Fisher) was added in excess to quantify bound CD69. The plate was again washed for three times followed by the addition of a secondary horseradish peroxidase (HRP) coupled antibody (Cell Signaling). Afterward, a peroxidase substrate solution (TMB; GE Healthcare) was dispensed to the plate and after 15 min of incubation, the stop solution H2SO4 (2N) was added to each well. The absorbance at 540 nm was measured using an ELISA reader (Anthos). A non‐linear regression analysis was performed to calculate the binding constant of CD69 to S100‐tetramer assuming a 1:1 binding model of one S100A8/S100A9‐tetramer binding one CD69‐dimer.[26 ]
6
Comprehensive Antibody Resource for Cell Biology
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The 8d4 talin-1/-2 antibody was purchased from Sigma-Aldrich (mouse). The Cortactin (goat), Tks5 (rabbit), Arp2 (rabbit), and GAPDH (rabbit) antibodies were obtained from Santa Cruz Biotechnology, Inc. The moesin antibodies were purchased from BD (mouse) and Cell Signaling Technology. The ezrin antibodies were obtained from Santa Cruz Biotechnology, Inc. (mouse) and Cell Signaling Technology (rabbit). The radixin and goat polyclonal internal talin antibodies were obtained from GeneTex (rabbit). The pan-phospho-threonine-ERM antibody was obtained from Cell Signaling Technology (rabbit). The chicken C-terminal (aa 639–815) NHE-1 antibody was a gift from D. Barber (University of California San Francisco, San Francisco, CA) and was used for Western blotting (Yan et al., 2001 (link)). The NHE-1 antibody used for immunofluorescence was obtained from Alpha Diagnostics (rabbit; NHE11-A). The TA205 talin head antibody was obtained from Enzo Life Sciences (mouse). The anti-His antibody was purchased from Thermo Fisher Scientific (mouse). The β1 integrin antibody (mouse) was purchased from BD. The FITC–anti-biotin antibody (mouse) was purchased from Jackson ImmunoResearch Laboratories, Inc. All Alexa Fluor secondary antibodies used were obtained from Molecular Probes. BCECF was obtained from Life Technologies.
7
In vitro PARylation and Phosphorylation Assays
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In vitro PARylation assays were performed as described previously (Kong et al., 2021 (link); Yao et al., 2021 (link)). Briefly, 500 ng HIS-FonPARP1 were incubated in a 50 μL PARylation buffer (50 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl2, pH8.0) with 0.2 mM NAD+, 1× activated DNA (BPS Bioscience, San Diego, CA, USA), and 3 μg GST-FonKin4 at 26°C for 3 h. Subsequently, the samples were separated on 8% and 12.5% SDS-PAGE. PARylated proteins were detected via immunoblotting with anti-poly-ADPR antibody (Cat. #MABE1031, Sigma-Aldrich), anti-GST antibody (Cat. #A00865, GenScript) or anti-HIS antibody (Cat. #A00186, GenScript).
For in vitro phosphorylation-mediated self-PARylation assays, 4 μg HIS-FonPARP1 underwent prior in vitro phosphorylation by HIS-FonKin4-ST, as described above. Then, 50 μL PARylation buffer was then added, followed by incubation at 26°C for 30 min. The reaction was stopped by adding 4× SDS loading buffer, and the proteins were then separated on 8% and 12.5% SDS-PAGE. Phosphorylated proteins were detected by anti-phosphor Ser/Thr antibody. PARylated proteins detected through immunoblotting with streptavidin-HRP (Cat. #21126, Thermo Fisher Scientific, Waltham, MA, USA) for Biotin NAD+ or anti-HIS antibody (Cat. #A00186, GenScript).
For in vitro phosphorylation-mediated self-PARylation assays, 4 μg HIS-FonPARP1 underwent prior in vitro phosphorylation by HIS-FonKin4-ST, as described above. Then, 50 μL PARylation buffer was then added, followed by incubation at 26°C for 30 min. The reaction was stopped by adding 4× SDS loading buffer, and the proteins were then separated on 8% and 12.5% SDS-PAGE. Phosphorylated proteins were detected by anti-phosphor Ser/Thr antibody. PARylated proteins detected through immunoblotting with streptavidin-HRP (Cat. #21126, Thermo Fisher Scientific, Waltham, MA, USA) for Biotin NAD+ or anti-HIS antibody (Cat. #A00186, GenScript).
8
Purification of His-Tagged Proteins
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Cell pellet was resuspended in 14 mL lysis buffer (50 mM Tris-HCl pH8.0, 500 mM NaCl, 20 mM imidazole, 1%v/v Tween20, 10%v/v glycerol, lysozyme 1 mg mL−1, DNase 0.1 mg mL−1) with protease inhibitors. The suspension was incubated at 4 °C for 30 min with stirring. Lysate was sonicated with Sonic Dismembrator (Fisher Scientific, 30% output, 3 min, 1 s off, 1 s on) in an ice-water bath, followed by centrifugation (20,000, 30 min) to collect the supernatant. Protino®Ni-NTA Agarose slurry (80 μL) was rinsed 3 times with wash buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole, 10%v/v glycerol) and incubated with the supernatant of cell lysate at 4 °C for 1 h. The mixture was loaded onto a Poly-Prep® Chromatography Column, washed 3 times with 5 mL wash buffer, and eluted with 200 μL elution buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole, 10%v/v glycerol) 5 times to obtain purified protein. Purified protein was exchanged to protein buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) using Amicon Ultra column, concentrated to 100 μL, and stored at −20 °C. For western blot detection of His-tagged proteins, the anti-His antibody from Thermo (Catalog number MA121315HRP) was used.
9
Detailed Reagents and Materials Protocol
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All routine reagents were procured from Sigma Aldrich, USA, unless otherwise mentioned. Oligonucleotides used in the study were procured from Integrated DNA Technologies (IDT, USA), Bioserve Biotechnologies (India), and Macrogen (South Korea). 96-well plates (MaxiSorp) were procured from Nunc, USA. Polyvinylidene fluoride (PVDF) membrane was obtained from Merck, USA. Bovine serum albumin (BSA) and nuclease free water (NFW) were procured from Himedia Laboratories, India. 3, 3′, 5, 5′-tetramethylbenzidine (TMB; BD OptEIA) was procured from BD Biosciences, USA. PCR master mix obtained from EmeraldAmp PCR master mix TakaraBio, Japan. Following antibodies are used in this study: anti-His antibody (Thermo Fisher Scientific, USA), anti-HIV-1 gp120 Env (ABLinc, USA), anti-HSA monoclonal antibody (R&D Systems, USA).
10
Purification and Binding Assay of RBS1
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The Rbs1 coding sequence was cloned by PCR into the HindIII and XhoI sites of pET28a to obtain pET-Rbs1. As a negative control, unrelated protein Avr-Pita was cloned into BamHI and SalI site of the same vector and designated as pET-AvrPita. The6XHIS-RBS1 or 6XHIS-Avr-Pita protein was induced in E. coli and purified using HIS-Select® Nickel Affinity Gel (Sigma) according to manufacturer’s instruction. Either purified 6XHIS-RBS1 or 6XHIS-Avr-Pita was incubated with beads containing polyuridylic, and polyguanylic ribohomopolymers as well as calf thymus single- and double stranded DNA purchased from Sigma. Incubation was done in 500 µl of buffer KHN (150 mM KCl, 20 mM HEPES pH7.9, 0.01% NP-40, complete protease inhibitors) for 10 min under rotation. Beads were then washed in KHN buffer 5 times and proteins retained in the beads were identified by Immunoblotting using anti-HIS antibody (Thermo Scientific).
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