Purified TACH-MAP30-LATA and MAP30 proteins were first quantified using Bradford method and then separated by SDS-PAGE in reducing and non-reducing conditions (with and without of β-mercaptoethanol). Protein samples were loaded into 12% gels SDS-PAGE and then transferred to a nitrocellulose membrane (1 h, 100 V). Following transfer, the membrane was blocked in Tris-buffered saline with Tween-20 containing 50 g/L skimmed milk for 2 h and then incubated with anti-6XHis tag antibody (Abcam, UK) for 2 h at room temperature. The membrane was washed three times with Tris-buffered saline (15 min each time) and then incubated with mouse anti-IGg antibody conjugated with alkaline phosphatase (Sigma, USA) for 2 h, washed again with Tris-buffered saline as described previously and finally developed with Western Blue® stabilized substrate (Promega, USA).
Anti 6x his tag antibody
Manufactured by Abcam
Sourced in United Kingdom
The Anti-6X His tag antibody is a highly specific antibody that recognizes the 6X His tag, which is a commonly used affinity tag for protein purification and detection. This antibody can be used to identify and detect proteins that have been tagged with the 6X His sequence.
Automatically generated - may contain errors
Lab products found in correlation
Maxibinding immunoplate, by SPL Life Sciences (2 mentions)
Polyclonal rabbit anti ha antibodies, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg h l hrp, by Abcam (2 mentions)
Western blue stabilized substrate, by Promega (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
C jun, by Abcam (1 mentions)
Octet htx system, by Molecular Devices (1 mentions)
Hbs ep buffer, by GE Healthcare (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Las software, by Leica (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Jetprime transfection reagent, by Polyplus Transfection (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting substrate, by Solarbio (1 mentions)
Molecular imager gel doc xr system, by Bio-Rad (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ibeam smart 488, by Toptica (1 mentions)
14 protocols using anti 6x his tag antibody
1
Western Blot Analysis of Recombinant Proteins
2
Ubiquitin Chain Conversion Assay
3
Interaction Analysis of mC3d and mFH/mFHR
4
Binding Kinetics of c-Jun and CAPER
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The binding kinetics of the peptides and full-length recombinant CAPER (R&D Systems, Minneapolis, MN) with c-Jun (Abcam, Cambridge, MA) were determined using biolayer interferometry (BLI) on the Octet HTX system (Pall ForteBio, Fremont, CA). Amine-Reactive 2nd Generation biosensors (cat# 18–5092, Pall ForteBio) were activated with 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride and N-hydroxysulfosuccinimide (EDC/NHS) (Amine Coupling Kit II, Cat# ACK-001-025, Sierra Sensors, Billerica, MA) and conjugated with an anti-6X His tag antibody (cat# ab9136, Abcam). The reaction was then quenched with 1M ethanolamine (Amine Coupling Kit II, Cat# ACK-001-025, Sierra Sensors). His-tagged recombinant c-Jun was then bound to the tips. The peptides were tested at a series of 2-fold dilutions including a blank buffer. The binding of full-length recombinant CAPER was also tested. Association and dissociation steps were performed for 900s each in 1X HBS-EP+ buffer (cat# BR100669, GE Healthcare Lifesciences, Pittsburgh, PA) supplemented with 450 mM NaCl (Sigma). The background was subtracted from each run, and kinetics data was analyzed using ForteBio’s Data Analysis Software version 10.0.3.1 using a 1:1 model.
5
Immunofluorescence Imaging of SARS-CoV-2 Proteins
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Human embryonic kidney 293T cells were grown in Dulbecco’s modified Eagle’s medium (Hyclone, South Logan, UT, USA) containing 10% foetal bovine serum (Gibco, NY, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) in a 5% CO2 incubator at 37°C. 293T cells were transfected with either the p-SARS-CoV-2 E/M or pcDNA3.1 (empty/mock) vector using jet PRIME transfection reagent (Polyplus, Illkirch, France). Cells were fixed in pre-cooled 4% paraformaldehyde, mobilised in 0.2% Triton X-100, and blocked by 10% goat serum in phosphate-buffered saline. Anti-6X His tag antibody (Abcam, Cambridge, UK) diluted at 1:50 was used as the primary antibody. After incubation, cells were washed and incubated with secondary antibodies [fluorescein isothiocyanate(FITC)-labelled goat anti-rabbit IgG] and 0.1% 4′,6-diamino-2-phenylindol (DAPI) at 37°C for 10 min. Fluorescent images were acquired using a Leica TCS SP8 confocal microscope with LAS software (Leica Biosystems, Wetzlar, Germany).
6
Protein Extraction and Analysis from HEK293T Cells
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HEK293T cell transformants were extracted using centrifugal force. The cells were then resuspended in M-PER™ Mammalian Protein Extraction Reagent containing protease inhibitor (Thermo Fisher Scientific, USA) at room temperature for 20 min. After the supernatant protein was collected by centrifugation, 4X protein loading buffer (GenScript Biotech, China) was added and denatured at 100 °C for 10 min. Proteins were resolved by 12% SDS-PAGE and transferred onto 0.45 nm pore polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using an eBlot™ L1 wet transfer (GenScript Biotech). Membranes were blocked and incubated with anti-6x His-tag antibody and secondary antibodies (goat anti-mouse; Abcam, UK) using eZwest Lite Automated Western Device (GenScript Biotech). Membranes were then incubated with ECL western blotting substrate (Solarbio) and captured using the Molecular Imager® Gel Doc™ XR System (Bio-Rad, USA).
7
Visualizing Membrane-Bound Proteins with SIM
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SLBs were
prepared as in “Formation of SLBs ” on glass microscope coverslips (Academy, 22 × 40 mm,
0.16–0.19 mm thick). The bilayers were incubated in 2% (w/v)
BSA solution for 1 h as a blocking step and then rinsed thoroughly
with liposome buffer. The primary antibody, anti6X His Tag antibody
(Abcam) was added to the bilayers in a 1:100 dilution in 0.2% (w/v)
BSA solution, and the bilayers were incubated at 4 °C overnight.
The bilayers were washed thoroughly with liposome buffer and then
incubated in goat antimouse IgG H&L (Alexa Fluor 488) secondary
antibody (Abcam) in a 0.2% (w/v) BSA solution for 1 h. The bilayers
were washed once in liposome buffer before imaging using SIM. The
wavelengths used for excitation were 561 nm (OBIS 561, Coherent) for
the lipid bilayers and 488 nm (iBEAM-SMART-488, Toptica) for the secondary
antibody.
prepared as in “
0.16–0.19 mm thick). The bilayers were incubated in 2% (w/v)
BSA solution for 1 h as a blocking step and then rinsed thoroughly
with liposome buffer. The primary antibody, anti6X His Tag antibody
(Abcam) was added to the bilayers in a 1:100 dilution in 0.2% (w/v)
BSA solution, and the bilayers were incubated at 4 °C overnight.
The bilayers were washed thoroughly with liposome buffer and then
incubated in goat antimouse IgG H&L (Alexa Fluor 488) secondary
antibody (Abcam) in a 0.2% (w/v) BSA solution for 1 h. The bilayers
were washed once in liposome buffer before imaging using SIM. The
wavelengths used for excitation were 561 nm (OBIS 561, Coherent) for
the lipid bilayers and 488 nm (iBEAM-SMART-488, Toptica) for the secondary
antibody.
8
Western Blot Analysis of His-Tagged Proteins
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HBP vacuoles were lysed, and the protein amount was estimated by Bradford protein assay. The samples were run on a 10% SDS–PAGE, transferred to nitrocellulose membranes and blocked with 5% skim milk in T–TBS. The membranes for HBP vacuoles were incubated overnight at 4 °C with Anti‐6X His tag antibody (abcam, UK, 1:1000 dilution). The blots were washed and incubated at room temperature for 1 h with Gt anti–Ms IgG (H + L) secondary antibodies (Invitrogen, 1: 5000 dilution). The membranes were washed and reacted with ECL STAR (DYNE, Korea). Immunofluorescent blots were developed by X‐ray film (AGFA).
9
Cholesterol-Binding EGFP-D4 Protein Production
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A plasmid containing hexahistidine (6xHis) tag-fused EGFP-D4 cloned with the pET28b vector was a gift from Toshihide Kobayashi, University of Strasbourg, Strasbourg, France. To increase the cholesterol affinity of the EGFP-D4 protein, three amino acids in the D4 were mutated (Y415A, D434W, and A463W), creating EGFP-D4YDA (25 (link)). Escherichia coli BL21 (DE3; Novagen) was transformed using the pET28b-EGFP-D4YDA plasmid and then cultured in Luria-Bertani broth (Formedium) containing 50 µg/mL kanamycin (Wako) at 37 °C until the optical density (OD600) reached 0.4. Then, 1 mM isopropyl β-d -thiogalactopyranoside (Wako) was added to the medium, and the cultures were induced for 3 h at 37 °C; 6xHis-tagged EGFP-D4YDA protein was purified using Ni-NTA Agarose (QIAGEN) and concentrated using an ultrafiltration column (Amicon Ultra-15 10K; Merck Millipore), according to the manufacturers’ instructions. The protein was analyzed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis with 0.1% Coomassie brilliant blue staining and Western blotting with an anti-6xHis tag antibody (Abcam).
10
Recombinant PLA2 Visualization and Activity
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To visualize recombinant PLA2s, the protein samples were electrophoresed in 12% sodium dodecyl sulfate– polyacrylamide gel, and were either stained using Coomassie blue or were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; EMD Millipore, USA). The membrane was then probed with anti- 6X His tag antibody (abcam, UK). After incubating the membrane with goat F(ab')2 Anti-mouse IgG (abcam) conjugated to alkaline phosphatase, the blot was developed using the BCIP/NBT chromogenic substrate solution (SurModics, Eden Prairie, USA) as specified by the manufacturer. The quantification of band intensity was carried out using the densitometry software (Total Lab 1.01; Nonlinear Dynamics Ltd.).
PLA2 activity was measured using the sPLA2 Assay Kit (Cayman Chemical, USA) according to the manufacturer’s instructions. The absorbance change at 37°C and 414 nm of wavelength was monitored by a spectrophotometer (OPTIZEN POP, Mecasys, Korea) after addition of enzyme solution. One unit (U) of PLA2 activity was defined as the amount of PLA2 able to hydrolyze 1 μmol of diheptanoyl thio-phosphatidylcholine in one minute.
PLA2 activity was measured using the sPLA2 Assay Kit (Cayman Chemical, USA) according to the manufacturer’s instructions. The absorbance change at 37°C and 414 nm of wavelength was monitored by a spectrophotometer (OPTIZEN POP, Mecasys, Korea) after addition of enzyme solution. One unit (U) of PLA2 activity was defined as the amount of PLA2 able to hydrolyze 1 μmol of diheptanoyl thio-phosphatidylcholine in one minute.
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