In total, 2 × 106 CD8-CD3ζ wt or IRAP ko cells were stained with biotinylated mouse anti-human CD8α (OKT8, Thermo Fisher Scientific, diluted 1/100) or mouse anti-human CD3ε (OKT3, Biolegend, diluted 1/100) at 4 °C for 30 min. Both antibodies were biotinylated with the EZ-Link® Sulfo-NHS-SS Biotinylation Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. After washing the unbound antibodies, cells were incubated for 15 min at 37 °C to induce endocytosis. The remaining antibody on the cell surface was blocked with streptavidin-FITC (Biolegend, diluted 1/100) for 20 min at 4 °C. After washing the unbound streptavidin, cells were re-incubated at 37 °C for 15, 30 and 60 min. The increased MFI relative to time point 0 corresponds to the amount of recycled CD3ε or CD3ζ.
Ez link sulfo nhs ss biotinylation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EZ-Link® Sulfo-NHS-SS Biotinylation Kit is a reagent kit used for the reversible biotinylation of proteins and other biomolecules. The kit contains Sulfo-NHS-SS-Biotin, a water-soluble, cleavable biotinylation reagent, and other necessary components for the biotinylation process.
Automatically generated - may contain errors
Lab products found in correlation
Pcsk9, by R&D Systems (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Amicon ultra 2 centrifugal unit, by Thermo Fisher Scientific (2 mentions)
Streptavidin fitc, by BioLegend (1 mentions)
Sypro ruby protein gel stain, by Thermo Fisher Scientific (1 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Gene synthesis, by GenScript (1 mentions)
Protein a sepharose cl 4b, by GE Healthcare (1 mentions)
Jetprime reagent, by Polyplus Transfection (1 mentions)
5 protocols using ez link sulfo nhs ss biotinylation kit
1
Investigating CD3 Recycling Dynamics
2
Antibody Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies were expressed by stably transfected Chinese hamster ovary (CHO)-S cell line and purified using Talon® Metal Affinity Resins (Clontech Laboratories, Inc., Mountain View, CA, USA), for BsAb A7/adalimumab and E2/adalimumab, or protein A Sepharose CL-48 resin (GE Healthcare, Chicago, IL, USA) for A7 scFv-Fc and adalimumab scFv-Fc. Antibodies were biotinylated using an EZ-Link Sulfo-NHS-SS Biotinylation Kit (ThermoFisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's protocol. Antibody purity was determined by SDS-PAGE via SYPRO Ruby Protein Gel Stain (Life Technologies, Carlsbad, CA, United States).
3
Production and Purification of Anti-ICAM-1 and Anti-TNF DVD Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequences of the variable regions of anti‐human ICAM‐1 antibody and human TNF have been previously described 20 , 21 . Sequence data management was performed using Serial Cloner 2.6. Variable sequences were generated by gene synthesis (GenScript) and combined into various constructs using overlapping‐extension polymerase chain reaction (PCR) 22 . The PCR products were cloned into the AbVec‐hIgG1 and AbVec‐hIgK vectors 23 using the restriction sites Age I–Sal I and Age I–Bsi WI, respectively. Clones were sequence‐verified prior to protein expression.
Twenty‐four hours before transfection, vectors encoding the heavy and light chains of the DVD antibody were transfected into HEK 293T cells in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 0.5 mg/ml of Geneticin. Transfection was performed with JetPrime reagent (Polyplus) according to the manufacturer's protocol. The antibodies were purified from the supernatant via affinity chromatography using protein A–Sepharose CL‐4B (GE Healthcare). DVD antibodies were biotinylated using an EZ‐Link Sulfo‐NHS‐SS biotinylation kit (Thermo‐Fisher Scientific) according to the manufacturer's protocol.
Twenty‐four hours before transfection, vectors encoding the heavy and light chains of the DVD antibody were transfected into HEK 293T cells in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 0.5 mg/ml of Geneticin. Transfection was performed with JetPrime reagent (Polyplus) according to the manufacturer's protocol. The antibodies were purified from the supernatant via affinity chromatography using protein A–Sepharose CL‐4B (GE Healthcare). DVD antibodies were biotinylated using an EZ‐Link Sulfo‐NHS‐SS biotinylation kit (Thermo‐Fisher Scientific) according to the manufacturer's protocol.
4
Biotinylation of recombinant PCSK9 protein
5
Biotinylation of recombinant PCSK9 protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before performing ProtoArray microarrays, recombinant (r) PCSK9 (carrier-free; R&D Systems; Minneapolis, MN) was biotinylated using the EZ Link Sulfo-NHS-SS Biotinylation kit (Pierce Thermo Scientific; Rockford, IL). First, to exchange buffers, 10 μg of rPCSK9 was added to a Zeba™ spin desalting column that has been pre-equilibrated according to the manufacturer’s protocol. Centrifugation was performed at 1000 × g for 2 minutes, and the resulting flow-through that contained the rPCSK9 protein in PBS was used in the next step. Sulfo-NHS-SS-Biotin was prepared by dissolving in ultrapure water at the concentration of 10 mM. Biotinylation of rPCSK9 was done with an at least 40-fold molar excess of Sulfo-NHS-SS-Biotin for 1 hour at room temperature. Filtering through another pre-equilibrated Zeba™ spin desalting column was performed as described above to remove excess biotin. Alternatively, excess biotin was removed by filtering through a 3 kDa Amicon ultra-2 centrifugal unit (Fisher Scientific; Pittsburg, PA). Protein concentrations were measured using a NanoDrop 2000 (Fisher Scientific). A HABA assay (reagents provided with the Biotinylation kit) was used to measure biotinylation levels. Calculations for the HABA assay were performed according to the protocol provided with the assay.
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!