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Ez link micro nhs peg4 biotinylation kit

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The EZ-Link Micro NHS-PEG4-Biotinylation Kit is a laboratory product designed for the biotinylation of proteins, peptides, and other biomolecules. The kit provides a convenient solution for adding a biotin label to target molecules, enabling their detection and purification in various downstream applications.

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Lab products found in correlation

Octet red biosensor, by Molecular Devices (3 mentions) Amicon ultra centrifugal filter, by Merck Group (1 mentions) Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions) Expi293f cells, by Thermo Fisher Scientific (1 mentions) Ni nta agarose, by Thermo Fisher Scientific (1 mentions) Facsaria 2, by BD (1 mentions) Streptavidin alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Streptavidin pe, by Thermo Fisher Scientific (1 mentions) Streptavidin apc, by BD (1 mentions) Ovalbumin (ova), by Merck Group (1 mentions) Human fc block, by BD (1 mentions) Anti cd20 pecy7, by BD (1 mentions) Cd19 microbeads, by Miltenyi Biotec (1 mentions) Human trustain fcx, by BioLegend (1 mentions) Zeba desalt spin column, by Thermo Fisher Scientific (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Cd hybridoma medium, by Thermo Fisher Scientific (1 mentions) Isostrip mouse monoclonal antibody isotyping kit, by Merck Group (1 mentions)

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EZ-Link NHS-PEG4 Biotinylation Kit (15 citations) Biotinylation kit (13 citations) EZ-Link micro NHS-PEG4 (2 citations)

16 protocols using ez link micro nhs peg4 biotinylation kit

1

Production and Purification of SARS-CoV-2 Spike Protein

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Recombinant soluble spike (S) protein derived from SARS-CoV-2 was expressed as previously described35 (link). In brief, a mammalian cell codon-optimized nucleotide sequences coding for the soluble version of S (GenBank: MN908947.3, amino acids 1-1,213) including a C-terminal thrombin cleavage site, T4 fold trimerization domain and hexahistidine tag was cloned into the mammalian expression vector pCAGGS. The S protein sequence was modified to remove the polybasic cleavage site (RRAR to A) and two stabilizing mutations were introduced (K986P and V987P, wild-type numbering). Recombinant proteins were produced in Expi293F cells (Thermo Fisher Scientific) by transfection with purified plasmid using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Supernatants from transfected cells were collected 3 days after transfection, and recombinant proteins were purified using Ni-NTA agarose (Thermo Fisher Scientific), then buffer-exchanged into PBS and concentrated using Amicon Ultra centrifugal filters (MilliporeSigma). For flow cytometry staining, recombinant S was labeled with Alexa Fluor 7647-NHS ester or biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation Kit (Thermo Fisher Scientific); excess Alexa Fluor 647 and biotin were removed using 7-kDa Zeba desalting columns (Thermo Fisher Scientific).
Kim W., Zhou J.Q., Sturtz A.J., Horvath S.C., Schmitz A.J., Lei T., Kalaidina E., Thapa M., Alsoussi W.B., Haile A., Klebert M.K., Suessen T., Parra-Rodriguez L., Mudd P.A., Middleton W.D., Teefey S.A., Pusic I., O’Halloran J.A., Presti R.M., Turner J.S, & Ellebedy A.H. (2021). Germinal centre-driven maturation of B cell response to SARS-CoV-2 vaccination. bioRxiv.
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2

Influenza Virus Hemagglutinin Staining

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For ELISPOT, we either used the TIV vaccine (diluted 1:20) or recombinant H1 HA protein derived from the pH1N1 (A/California/06/2009) influenza virus. HA was obtained from the Biodefense and Emerging Infections (BEI) research repository. For HA staining, we used the HA1 subunit from H1 HA of A/California/06/2009 (H1N1) that was expressed in 293 T cells. HA molecules were biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation kit (Thermo-Fisher). Excess biotin was removed using the Zeba Spin Desalting Columns (Pierce).
Ellebedy A.H., Jackson K.J., Kissick H.T., Nakaya H.I., Davis C.W., Roskin K.M., McElroy A.K., Oshansky C.M., Elbein R., Thomas S., Lyon G.M., Spiropoulou C.F., Mehta A.K., Thomas P.G., Boyd S.D, & Ahmed R. (2016). Defining antigen-specific plasmablast and memory B cell subsets in blood following viral infection and vaccination of humans. Nature immunology, 17(10), 1226-1234.
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3

Biotinylated GP Competitive Binding Assay

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Biotinylated GP or GPΔmuc (EZ-link® Micro NHS-PEG4-Biotinylation Kit, Thermo Scientific #21955) (1 μg/mL) was immobilized onto streptavidin-coated biosensor tips (ForteBio #18-5019) for 2 minutes. After measuring the baseline signal in kinetics buffer (KB: 1× PBS, 0.01% BSA and 0.002% Tween 20) for two minutes, biosensor tips were immersed into the wells containing primary antibody at a concentration of 100 μg/mL for 10 minutes. Biosensors then were immersed into wells containing competing mAbs at a concentration of 100 μg/mL for 5 minutes. The percent binding of the competing mAb in the presence of the first mAb was determined by comparing the maximal signal of competing mAb applied after the first mAb complex to the maximal signal of competing mAb alone. MAbs were judged to compete for binding to the same site if maximum binding of the competing mAb was reduced to <30% of its un-competed binding. MAbs were considered non-competing if maximum binding of the competing mAb was >70% of its un-competed binding. A level of 30-70% of its un-competed binding was considered intermediate competition.
Flyak A.I., Ilinykh P.A., Murin C.D., Garron T., Shen X., Fusco M.L., Hashiguchi T., Bornholdt Z.A., Slaughter J.C., Sapparapu G., Klages C., Ksiazek T.G., Ward A.B., Saphire E.O., Bukreyev A, & Crowe JE J.r. (2015). Mechanism of Human Antibody-Mediated Neutralization of Marburg Virus. Cell, 160(5), 893-903.
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4

Biotinylation of Bovine Lactadherin

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Bovine lactadherin was biotinylated using the EZ-Link Micro NHS-PEG
4-Biotinylation kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States) according to instructions from the manufacturer. Fifty-fold excess NHS-PEG
4was added to lactadherin and the mixture was incubated for 30 minutes at room temperature. Zeba spin desalting columns were used to remove unreacted biotin. Aliquots of 2 µM biotinylated lactadherin were frozen at −70°C until use.
Thulin Å., Yan J., Åberg M., Christersson C., Kamali-Moghaddam M, & Siegbahn A. (2018). Sensitive and Specific Detection of Platelet-Derived and Tissue Factor–Positive Extracellular Vesicles in Plasma Using Solid-Phase Proximity Ligation Assay. TH Open: Companion Journal to Thrombosis and Haemostasis, 2(3), e250-e260.
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5

Influenza ELISpot Protocol with Recombinant HA

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For ELISpot, plates were coated with QIV, tetanus/diphtheria vaccine
(Grifols), or recombinant hemagglutinin (HA) proteins derived from the pandemic
H1N1 (A/Michigan/45/2015), H3N2 (A/Singapore/INFIMH-16-0019/2016),
B/Yamagata/16/88-like lineage (B/Phuket/3073/2013), or B/Victoria/2/87-like
lineage (B/Brisbane/60/2008) influenza viruses. HA proteins were expressed in a
baculovirus expression system as described previously29 (link). For flow cytometry staining,
recombinant HA from A/Michigan/45/2015 (a.a.18–529),
A/Singapore/INFIMH-16-0019/2016 (a.a.17–529), and B/Colorado/06/2017
(a.a. 18–546) expressed in 293F cells were purchased from Immune
Technology and biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation Kit
(Thermo Fisher); excess biotin was removed using 7-kDa Zeba desalting columns
(Pierce).
Turner J.S., Zhou J.Q., Han J., Schmitz A.J., Rizk A.A., Alsoussi W.B., Lei T., Amor M., McIntire K.M., Meade P., Strohmeier S., Brent R.I., Richey S.T., Haile A., Yang Y.R., Klebert M.K., Suessen T., Teefey S., Presti R.M., Krammer F., Kleinstein S.H., Ward A.B, & Ellebedy A.H. (2020). Human germinal centres engage memory and naïve B cells after influenza vaccination. Nature, 586(7827), 127-132.
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6

Competition Binding Assay for EBOV GP

Cited 1 time
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Competition-binding studies using biolayer interferometry and biotinylated EBOV GP (EZ-link® Micro NHS-PEG4-Biotinylation Kit, Thermo Scientific #21955) (5 μg/mL) were performed on an Octet RED biosensor (ForteBio Menlo Park, CA), as described previously27 (link). In brief, the antigen was immobilized onto streptavidin-coated biosensor tips. After a brief washing step, biosensor tips were immersed first into the wells containing primary antibody at a concentration of 100 μg/mL and then into the wells containing competing mAbs at a concentration of 100 μg/mL. The percent binding of the competing mAb in the presence of the first mAb was determined by comparing the maximal signal of competing mAb applied after the first mAb complex to the maximal signal of competing mAb alone.
Flyak A.I., Kuzmina N., Murin C.D., Bryan C., Davidson E., Gilchuk P., Gulka C.P., Ilinykh P.A., Shen X., Huang K., Ramanathan P., Turner H., Fusco M.L., Lampley R., Kose N., King H., Sapparapu G., Doranz B.J., Ksiazek T.G., Wright D.W., Saphire E.O., Ward A.B., Bukreyev A, & Crowe JE J.r. (2018). BROADLY NEUTRALIZING ANTIBODIES FROM HUMAN SURVIVORS TARGET A CONSERVED SITE IN THE EBOLA VIRUS GLYCOPROTEIN HR2/MPER REGION. Nature microbiology, 3(6), 670-677.
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7

Competition Binding Assay for EBOV GP

Cited 1 time
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Competition-binding studies using biolayer interferometry and biotinylated EBOV GP (EZ-link® Micro NHS-PEG4-Biotinylation Kit, Thermo Scientific #21955) (5 μg/mL) were performed on an Octet RED biosensor (ForteBio Menlo Park, CA), as described previously27 (link). In brief, the antigen was immobilized onto streptavidin-coated biosensor tips. After a brief washing step, biosensor tips were immersed first into the wells containing primary antibody at a concentration of 100 μg/mL and then into the wells containing competing mAbs at a concentration of 100 μg/mL. The percent binding of the competing mAb in the presence of the first mAb was determined by comparing the maximal signal of competing mAb applied after the first mAb complex to the maximal signal of competing mAb alone.
Flyak A.I., Kuzmina N., Murin C.D., Bryan C., Davidson E., Gilchuk P., Gulka C.P., Ilinykh P.A., Shen X., Huang K., Ramanathan P., Turner H., Fusco M.L., Lampley R., Kose N., King H., Sapparapu G., Doranz B.J., Ksiazek T.G., Wright D.W., Saphire E.O., Ward A.B., Bukreyev A, & Crowe JE J.r. (2018). BROADLY NEUTRALIZING ANTIBODIES FROM HUMAN SURVIVORS TARGET A CONSERVED SITE IN THE EBOLA VIRUS GLYCOPROTEIN HR2/MPER REGION. Nature microbiology, 3(6), 670-677.
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8

Influenza Virus Hemagglutinin Staining

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For ELISPOT, we either used the TIV vaccine (diluted 1:20) or recombinant H1 HA protein derived from the pH1N1 (A/California/06/2009) influenza virus. HA was obtained from the Biodefense and Emerging Infections (BEI) research repository. For HA staining, we used the HA1 subunit from H1 HA of A/California/06/2009 (H1N1) that was expressed in 293 T cells. HA molecules were biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation kit (Thermo-Fisher). Excess biotin was removed using the Zeba Spin Desalting Columns (Pierce).
Ellebedy A.H., Jackson K.J., Kissick H.T., Nakaya H.I., Davis C.W., Roskin K.M., McElroy A.K., Oshansky C.M., Elbein R., Thomas S., Lyon G.M., Spiropoulou C.F., Mehta A.K., Thomas P.G., Boyd S.D, & Ahmed R. (2016). Defining antigen-specific plasmablast and memory B cell subsets in blood following viral infection and vaccination of humans. Nature immunology, 17(10), 1226-1234.
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9

Influenza ELISpot Protocol with Recombinant HA

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For ELISpot, plates were coated with QIV, tetanus/diphtheria vaccine
(Grifols), or recombinant hemagglutinin (HA) proteins derived from the pandemic
H1N1 (A/Michigan/45/2015), H3N2 (A/Singapore/INFIMH-16-0019/2016),
B/Yamagata/16/88-like lineage (B/Phuket/3073/2013), or B/Victoria/2/87-like
lineage (B/Brisbane/60/2008) influenza viruses. HA proteins were expressed in a
baculovirus expression system as described previously29 (link). For flow cytometry staining,
recombinant HA from A/Michigan/45/2015 (a.a.18–529),
A/Singapore/INFIMH-16-0019/2016 (a.a.17–529), and B/Colorado/06/2017
(a.a. 18–546) expressed in 293F cells were purchased from Immune
Technology and biotinylated using the EZ-Link Micro NHS-PEG4-Biotinylation Kit
(Thermo Fisher); excess biotin was removed using 7-kDa Zeba desalting columns
(Pierce).
Turner J.S., Zhou J.Q., Han J., Schmitz A.J., Rizk A.A., Alsoussi W.B., Lei T., Amor M., McIntire K.M., Meade P., Strohmeier S., Brent R.I., Richey S.T., Haile A., Yang Y.R., Klebert M.K., Suessen T., Teefey S., Presti R.M., Krammer F., Kleinstein S.H., Ward A.B, & Ellebedy A.H. (2020). Human germinal centres engage memory and naïve B cells after influenza vaccination. Nature, 586(7827), 127-132.
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10

Isolation of SARS-CoV-2 S-Protein-Binding Memory B Cells

Cited 2 times
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S-protein (adr serotype) expressed and purified from Chinese hamster ovary (CHO) cells (ProSpec) and ovalbumin (Sigma-Aldrich) were biotinylated using EZ-Link™ Micro NHS-PEG4-Biotinylation kit (Thermo Fisher Scientific). S-protein-PE and S-protein-APC were prepared by incubating 2–3 μg of biotin-S-protein with streptavidin-PE (eBioscience) or streptavidin-APC (BD Biosciences) in PBS respectively overnight at 4°C in the dark. ovalbumin-Alexa Fluor 488 was generated by incubating biotin-ovalbumin with streptavidin-Alexa Fluor 488 (Thermo Fisher Scientific).
B cell purification, labeling, and sorting were as previously described (Escolano et al., 2019 (link); Robbiani et al., 2017 (link); Tiller et al., 2008 (link); von Boehmer et al., 2016 (link)). Briefly, PBMCs were thawed and washed with RPMI medium at 37°C. B lymphocytes were positively selected using CD19 MicroBeads (Miltenyi Biotec) followed by incubation with human Fc block (BD Biosciences) and anti-CD20-PECy7 (BD Biosciences), anti-IgG-Bv421 (BD Biosciences), S-protein-PE at 10 μg/ml, S-protein-APC at 10 μg/ml, and ovalbumin-Alexa Fluor 488 at 10 μg/ml at 4°C for 20 minutes. Single CD20+ IgG+ S-protein-PE+ S-protein-APC+ Ova-Alexa Fluor 488− memory B cells were sorted into 96-well plates using a FACSAriaII (Becton Dickinson) and stored at −80°C.
Wang Q., Michailidis E., Yu Y., Wang Z., Hurley A.M., Oren D.A., Mayer C.T., Gazumyan A., Liu Z., Zhou Y., Schoofs T., Yao K.H., Nieke J.P., Wu J., Jiang Q., Zou C., Kabbani M., Quirk C., Oliveira T., Chhosphel K., Zhang Q., Schneider W.M., Jahan C., Ying T., Horowitz J., Caskey M., Jankovic M., Robbiani D.F., Wen Y., de Jong Y.P., Rice C.M, & Nussenzweig M.C. (2020). A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations. Cell host & microbe, 28(2), 335-349.e6.
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