LAD2 cells (3 × 105) were incubated with Spike-RBD protein (5 μg/mL, Genscript, Z03483) in adherent buffer (1 mM CaCl2, 2 mM MgCl2 and 5% BSA, pH 7.4) for 1 h at 4 °C. The cells were then fixed with 4% paraformaldehyde (Sigma–Aldrich) for 30 min at room temperature and stained with anti-His-tag antibodies (Abmart, M30111S). Subsequently, the cells were stained with goat anti-mouse Alexla Fluor 488-conjugated secondary antibodies (Invitrogen, A11001), and were detected with flow cytometry (BD Accuri C6) and analyzed with the FlowJo 7.6.1 software. In some experiments, LAD2 cells were prior-blocked with anti-ACE2 antibody (5 μg/mL, R&D Systems, AF933) for 1 h at 37 °C before the incubation with Spike-RBD protein.
Anti his tag antibody
Manufactured by Abmart
Sourced in China
The Anti-His Tag antibody is a laboratory reagent used to detect and purify proteins that have been engineered to contain a histidine (His) tag. The antibody specifically binds to the His tag, which is a sequence of amino acids commonly added to recombinant proteins to facilitate their identification and purification.
Automatically generated - may contain errors
Lab products found in correlation
Spike rbd protein, by GenScript (2 mentions)
Goat anti mouse alexla fluor 488 conjugated secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
G8772, by Merck Group (2 mentions)
Fastprep 24, by MP Biomedicals (2 mentions)
Af933, by R&D Systems (1 mentions)
Accuri c6, by BD (1 mentions)
Anti ace2 antibody, by R&D Systems (1 mentions)
P30266, by Abmart (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Genegnome hr system, by Syngene (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
5 protocols using anti his tag antibody
1
Spike-RBD Protein Binding to LAD2 Cells
2
Spike Protein Binding to LAD2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LAD2 cells (3×10 5 ) were incubated with Spike-RBD protein (5 μg/mL, Genscript, Z03483) in adherent buffer (1mM CaCl 2 , 2mM MgCl 2 and 5% BSA, pH 7.4) for 1h at 4 °C. The cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature and stained with anti-His-tag antibodies (Abmart, M30111S). Subsequently, the cells were stained with goat anti-mouse Alexla Fluor 488-conjugated secondary antibodies (Invitrogen, A11001), and were detected with flow cytometry. In some experiments, LAD2 cells were prior-treated with 0.25% trypsin (without EDTA) for 10 min at 37 °C or prior-blocked with anti-ACE2 antibody (5 μg/mL, R&D Systems, AF933) for 2 h at 37 °C before the incubation with Spike-RBD protein.
3
Yeast Expression and Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transformants were grown in YG mediums (2% yeast extract, 4% glucose, pH 6.0) at 30 °C, 220 rpm for 72 h. One milliliter of cultures was harvested and centrifuged for 10 min at 5000 rpm to detect the secretory or intracellular expression of enzymes by western blot. To prepare lysate samples, cells were suspended in 1 mL lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate), and then disrupted by a bead-beater (FastPrep-24, MP, California, USA) at 6 m/s for 2 min with 400 μL acid-washed glass beads (G8772, Sigma-Aldrich, Missouri, USA). Western blots were carried out using an Anti-His Tag antibody (M30111, Abmart, Shanghai, China) and a horseradish peroxidase-conjugated goat-anti-mouse secondary antibody (074–1806, KPL, USA) as described previously [18 (link)].
4
Acetyllysine Antibody-Based Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All strains (Supplementary Table S1 ), plasmids (Supplementary Table S2 ), primers (Supplementary Table S3 ), and culture media used in this study are listed in the Supplemental Information. The pan anti-acetyllysine antibody used in this study was the same as that described in (23 (link)), and the anti-His-tag antibody was purchased from Abmart, Shanghai, China.
5
Western Blot Analysis of FIM-1Δu Transformants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pZP32 and pZP33 were transformed into FIM-1∆U. Transformants were grown in YG medium and 3 × 108 cells were collected after indicated times. Cells were washed and resuspended in 400 μL lysis buffer (50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate) supplemented with protease inhibitors cocktail (05892970001, Roche Applied Science). Cells were mixed with 400 μL acid-washed glass beads (G8772, Sigma-Aldrich, Missouri, USA) and processed by a bead-beater (FastPrep-24, MP, California, USA) at 6 m/s for 2 min. Lysate was centrifuged at 13,200 rpm for 20 min at 4°. 100 μL Supernatant was supplemented with 25 μL 5XSDS PAGE loading buffer (150 mM Tris–HCl (pH 7.0), 12% SDS, 6% 2-mercaptoethanol, 30% glycerol (V/V), 0.05% Coomassie Brilliant Blue G-250) and boiled. 10 μL Samples were subjected to Western blot assay [28 (link)]. Anti-His Tag antibody (1:5000 dilution) (M30111, Abmart, Shanghai, China), anti-histone H3.1 antibody (1:3000 dilution) (P30266, Abmart) and horseradish peroxidase conjugated goat-anti-mouse secondary antibody (1:3000 dilution) (074–1806, KPL, USA) were used in the Western blot. The blots were visualized by ECL prime Western blotting detection reagent (RPN2232, GE Healthcare, Illinois, USA) and scanned by GeneGnome HR system (Syngene, Cambridge, UK).
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!