Vero cells were seated on a 12-well plate at a cell density of 2 × 105 cells/well. Cells were infected with SARS-CoV-2 at an MOI of 1 and incubated at 37°C for 24 h. The cultured cells were subjected to centrifugation at 1,600 × g for 5 min. The supernatant was removed, and the cells were lysed using 1 × SDS sample buffer. The lysed cells were boiled at 95°C for 3 min, sonicated using Bioruptor II (BM Equipment, Tokyo, Japan), and centrifuged at 16,400 × g for 3 min. The samples were loaded onto a 10% TGX FastCast Acrylamide gel (Bio-Rad, CA, USA) and electrophoresed. Then, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with PBS containing 0.05% tween-20 (PBS-T; Nacalai Tesque) and 5% skimmed milk (Nacalai Tesque) and reacted with rabbit anti-SARS-CoV-2 S1 (clone: SA39; Cell Engineering Corporation, Osaka, Japan) or mouse anti-S2 monoclonal antibodies (GeneTex, CA, USA). The membrane was washed with PBS-T twice and reacted with HRP-conjugated anti-mouse or rabbit IgG antibodies (Sigma-Aldrich). Protein bands were visualized using Chemi-Lumi One Ultra (Nacalai Tesque) and Amersham ImageQuant 800 (GE Healthcare, IL, USA).
Pbs t
Manufactured by Nacalai Tesque
Sourced in Japan
PBS-T is a buffer solution used in various laboratory techniques. It is a mixture of phosphate-buffered saline (PBS) and Tween-20, a non-ionic detergent. PBS-T is commonly used for washing and diluting samples in immunoassays, Western blotting, and other biochemical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Chemi lumi one ultra, by Nacalai Tesque (2 mentions)
Skimmed milk, by Nacalai Tesque (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hrp conjugated anti mouse or rabbit igg antibodies, by Merck Group (1 mentions)
Amersham imagequant 800, by GE Healthcare (1 mentions)
Tgx fastcast acrylamide gel, by Bio-Rad (1 mentions)
Goat anti mouse igg horseradish peroxidase hrp, by Merck Group (1 mentions)
Mouse anti β actin antibody, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Elisa pod substrate tmb kit, by Nacalai Tesque (1 mentions)
Nunc maxisorp 96 well immunoplates, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated anti mouse immunoglobulin, by Agilent Technologies (1 mentions)
Imark microplate reader, by Bio-Rad (1 mentions)
3 protocols using pbs t
1
SARS-CoV-2 Spike Protein Detection
2
Western Blot Analysis of FCoV Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Infected Fcwf-4, A72, MDCK, and DH82 cells were lysed in lysis buffer (100 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 1% Triton X-100) and centrifuged at 16,000 × g for 10 min at 4°C. Supernatants were then collected and mixed with 2× sample buffer (0.1 M Tris-HCl [pH 6.8], 4% sodium dodecyl sulfate [SDS], 20% glycerol, 0.004% bromophenol blue, 10% 2-mercaptoethanol). Next, the boiled samples were subjected to electrophoresis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Billerica, MA). The membranes were blocked in 3% skim milk–PBS containing 0.05% Tween 20 (PBS-T; Nacalai Tesque). The primary antibodies were mouse anti-FCoV-N monoclonal antibody (4D10) and mouse anti-β-actin antibody (Sigma-Aldrich), while goat anti-mouse IgG horseradish peroxidase (HRP; Sigma-Aldrich) was used as a secondary antibody. ChemiLumi One Ultra (Nacalai Tesque) was used for visualization (32 (link)).
3
SARS-CoV-2 Spike Protein ELISA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
His-tagged S1 spike protein of SARS-CoV-2 (100 μg) or 100 μg of His-tagged S2 spike protein of SARS-CoV-2 was immobilized on Nunc Maxisorp 96-well immunoplates (Cat# 439454, Thermo Fisher Scientific) at 1 μg/mL for 30 min at 37°C. After washing with phosphate buffered saline (PBS) containing 0.05% Tween 20 (PBS-T; Nacalai Tesque), wells were blocked with 1% bovine serum albumin (BSA) in PBS-T for 30 min at 37°C. The plates were incubated with primary antibodies followed by peroxidase-conjugated anti-mouse immunoglobulin (1:1000; Agilent Technologies, Santa Clara, CA, USA). Finally, enzymatic reactions were performed using an ELISA POD substrate TMB kit (Cat# 05298-80, Nacalai Tesque). The absorbance at 655 nm was measured using an iMark microplate reader (Bio-Rad Laboratories, Berkeley, CA).
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!