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Protein labelling kit

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The Protein Labelling Kit is a laboratory tool designed to enable the covalent attachment of a label to proteins. The kit includes reagents and protocols for performing this labelling process, which is commonly used in various analytical techniques and applications.

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Lab products found in correlation

Monolith nt 115 glass, by NanoTemper (2 mentions) Antibody labeling kit, by Thermo Fisher Scientific (1 mentions) Ez link micro sulfo nhs biotinylation kit, by Thermo Fisher Scientific (1 mentions) Cary eclipse fluorescence spectrophotometer, by Agilent Technologies (1 mentions) Ghost violet 510 viability dye, by Cytek Biosciences (1 mentions) Lsrii fortessa x 20 flow cytometer, by BD (1 mentions) Cd11b percpcy5.5 m1 70, by Cytek Biosciences (1 mentions) Sa bv421, by BioLegend (1 mentions) Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions) Fluorescence mounting medium, by Agilent Technologies (1 mentions) Axio observer, by Zeiss (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Afacscan, by BD (1 mentions)

Spelling variants of this product

Alexa Fluor™ 488 Protein Labelling Kit (5 citations) Alexa Fluor 488 Microscale Protein Labelling Kit (5 citations) Protein-X labelling kit (2 citations) Alexa Fluor 647 Protein Labelling Kit (2 citations) Alexa Fluor 568 protein labelling kit (2 citations) Alexa Fluor 532 protein labelling kit (2 citations)

11 protocols using protein labelling kit

1

Antibody Labeling Techniques

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Antibodies were conjugated to Alexa Fluor 647 using Antibody Labeling Kit (ThermoFisher), pHrodo iFL Green using Protein labelling kit (ThermoFisher) or Biotin using EZ-Link Micro Sulfo-NHS-Biotinylation kit (ThermoFisher) according to manufacturer’s instructions.
Panova V., Attig J., Young G.R., Stoye J.P, & Kassiotis G. (2020). Antibody-induced internalisation of retroviral envelope glycoproteins is a signal initiation event. PLoS Pathogens, 16(5), e1008605.
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2

Mapping FRET-based Protein Interactions

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MtbXthA was labelled with Oregon green 488 maleimide (MtbXthAOG) while BRCT domain of MtbLigA was labelled with Alexa flour 555 C2-maleimide (BRCTAF) using a protein labelling kit (Thermo Scientific). The degree of labelling was found to be three fluorophores per MtbXthA and two fluorophores per BRCT domain, respectively as determined by following the manufacturer's instructions. Interaction between the two labelled proteins was monitored by fluorescence resonance energy transfer (FRET). The donor MtbXthAOG (0.2 μM) and acceptor BRCTAF (2 μM) were incubated in 500 μl FRET buffer (50 mM HEPES–Na pH 8.0, 50 mM NaCl, 2 mM DTT, 8 mM MgCl2 and 0.5 mM EDTA) for 30 min on ice followed by measurement at room temperature using Cary Eclipse Fluorescence spectrophotometer (Agilent Technologies). Samples were excited at 488 nm and emission spectra were recorded from 495 to 600 nm. The complex disruption was tested by adding respective peptides and monitoring any change in the FRET efficiency. FRET efficiency (E) was calculated by; E = 1 – Fd’/Fd where Fd’ is the emission intensity of the donor in the presence of acceptor and Fd is the emission intensity of the donor alone. The peptides used in the study include (i) DGQ (DGQPSWSGKP) (ii) YDV (YDVAHVGFD) and (iii) Control peptide (IGQFDLFGV)
Khanam T., Afsar M., Shukla A., Alam F., Kumar S., Soyar H., Dolma K., A., Pasupuleti M., Srivastava K.K., Ampapathi R.S, & Ramachandran R. (2020). M. tuberculosis class II apurinic/ apyrimidinic‐endonuclease/3′‐5′ exonuclease (XthA) engages with NAD+-dependent DNA ligase A (LigA) to counter futile cleavage and ligation cycles in base excision repair. Nucleic Acids Research, 48(8), 4325-4343.
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3

Fluorescent Ligand Binding Assay for Trypanosoma

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Hb and BSA were labelled with Alexa Fluor 488 using a protein labelling kit (Thermo Fisher Scientific). T. congolense WG81 epimastigote-containing cultures (generated as above) were grown on coverslips overnight in serum-free Cunningham’s media supplemented with 5 mg/ml BSA, 1 mM hypoxanthine and 0.16 mM thymidine. Incubation in serum-free media was required to remove competing Hb ligand from the media. Coverslips were moved onto poly-l-lysine slides and incubated with no ligand, 10 nM Hb-488 or 10 nM-BSA at 27°C for 4 hr. At 2 hr post-addition of ligand, 2 µM protease inhibitor FMK-024 was added. Cells were fixed in 4% paraformaldehyde for 30 min at room temperature, washed 3x in PBS, stained with 1 µg/ml DAPI for 5 min and mounted. Microscopy was carried out as above.
Lane-Serff H., MacGregor P., Peacock L., Macleod O.J., Kay C., Gibson W., Higgins M.K, & Carrington M. (2016). Evolutionary diversification of the trypanosome haptoglobin-haemoglobin receptor from an ancestral haemoglobin receptor. eLife, 5, e13044.
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4

Multicolor Flow Cytometry Immunophenotyping

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Purified monoclonal antibody was directly conjugated to Alexa Fluor-488 by protein labelling kit (ThermoFisher Scientific) according to the manufacturer's protocol. Splenocytes were isolated from NOD mice, red blood cells lysed and resuspended at in complete DMEM at 6 × 106 cells ml−1. p63 or OVA141–160 was added to the media at a final concentration of 40 μM and the cells were incubated for 1.5 h at 37 °C with 5% CO2. Cells were collected incubated in 2.4G2 for 10 min on ice and stained at 1:100 dilution for 30 min at 4 °C with FS1-AF488 and antibodies against CD4-BUV395 (GK1.5, BD Biosciences), CD8α-APC-ef780 (53–6.7, eBioscience), CD3ɛ-BV650 (145-2C11, BD Biosciences), B220-PE (RA3-6B, Tonbo), CD11c-PE-Cy (N418, eBioscience), CD11b-PerCp-Cy5.5 (M1/70, Tonbo), F4/80-APC (BM8.1, Tonbo), IAg7-biotin (10.2-16, BioXcell), SA-BV421 (BioLegend) and dead cells were gated out using Ghost violet 510 viability dye (Tonbo), and run on a LSRII Fortessa X-20 flow cytometer (Becton Dickinson) and analyzed using FlowJo software (v10).
Spanier J.A., Frederick D.R., Taylor J.J., Heffernan J.R., Kotov D.I., Martinov T., Osum K.C., Ruggiero J.L., Rust B.J., Landry S.J., Jenkins M.K., McLachlan J.B, & Fife B.T. (2016). Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment. Nature Communications, 7, 11804.
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5

Fluorescent Labeling of Glycoproteins

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BSA, BSA-α-Gal, BSA-NAl, HSA, HSA-α-Gal and bTG were fluorescently labeled with Alexa Fluor 488 (AF 488) using the protein labelling kit (Thermo Fisher, Rockford, IL, US) according to the manufacturer’s instructions. Protein concentration was determined using the bicinchoninic acid protein assay kit (Life technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The endotoxin levels were determined by the limulus amebocyte lysate assay (Charles River Endosafe, Charleston, SC, USA) to less than 1 ng LPS/mg of protein in all protein preparations. The labeled proteins were stored at −20 °C and protected from light until further use.
Ristivojević M.K., Grundström J., Tran T.A., Apostolovic D., Radoi V., Starkhammar M., Vukojević V., Ćirković Veličković T., Hamsten C, & van Hage M. (2018). α-Gal on the protein surface affects uptake and degradation in immature monocyte derived dendritic cells. Scientific Reports, 8, 12684.
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6

GSDMD Ligand Binding Assay by MST

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His6-MBP-GSDMD was labelled with AlexaFluor-488 using the Molecular Probes protein labelling kit. Binding of inhibitors to GSDMD was evaluated using microscale thermophoresis (MST). Ligands (49 nM - 150 μM) were incubated with purified AlexaFluor-488-labeled protein (80 nM) for 30 min in assay buffer (20 mM HEPES at pH 7.4, 150 mM NaCl, 0.05% Tween 20). The sample was loaded into NanoTemper Monolith NT.115 glass capillaries and MST carried out using 20% LED power and 40% MST power. KD values were calculated using the mass action equation and NanoTemper software.
Hu J.J., Liu X., Xia S., Zhang Z., Zhang Y., Zhao J., Ruan J., Luo X., Lou X., Bai Y., Wang J., Hollingsworth L.R., Magupalli V.G., Zhao L., Luo H.R., Kim J., Lieberman J, & Wu H. (2020). FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation. Nature immunology, 21(7), 736-745.
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7

GSDMD Ligand Binding Assay by MST

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His6-MBP-GSDMD was labelled with AlexaFluor-488 using the Molecular Probes protein labelling kit. Binding of inhibitors to GSDMD was evaluated using microscale thermophoresis (MST). Ligands (49 nM - 150 μM) were incubated with purified AlexaFluor-488-labeled protein (80 nM) for 30 min in assay buffer (20 mM HEPES at pH 7.4, 150 mM NaCl, 0.05% Tween 20). The sample was loaded into NanoTemper Monolith NT.115 glass capillaries and MST carried out using 20% LED power and 40% MST power. KD values were calculated using the mass action equation and NanoTemper software.
Hu J.J., Liu X., Xia S., Zhang Z., Zhang Y., Zhao J., Ruan J., Luo X., Lou X., Bai Y., Wang J., Hollingsworth L.R., Magupalli V.G., Zhao L., Luo H.R., Kim J., Lieberman J, & Wu H. (2020). FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation. Nature immunology, 21(7), 736-745.
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8

GSDMD Activation and Bacterial Labeling

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Full-length and C-terminal GSDMD was labelled with AlexaFluor-488 using the Molecular Probes protein labelling kit. An aliquot of the labelled full-length protein was activated by incubating with active caspase-11 for 15 min at 37°C. L. monocytogenes expressing mCherry (a gift from J. Theriot, Stanford Medical School) were treated with PBS or with 500 nM AF488-labelled GSDMD that had been activated or not with caspase-11 or with GSDMD-CT for 30 min at 37°C. Bacteria were washed with 10 mM arginine in PBS for 10 min before fixation in 2% formalin in PBS. Slides were mounted with fluorescence mounting medium (Dako) and imaged using a fully motorized Axio Observer spinning disk microscope (Carl Zeiss Microimaging, Inc.) equipped with a cooled electron multiplication CCD camera with 512 × 512 resolution (Photometrics QuantEM, Tuscon, AZ) with excitation filters set at 405, 488, 561 and 640 nm and emission filter ranges of 430–475, 500–550, 589–625 and 680 nm long-pass, respectively. Images were analysed using SlideBook V5.0 (Intelligent Imaging Inc.) software. Third-dimensional image stacks were obtained along the z axis using the 63× oil immersion objective by acquiring sequential optical planes spaced 0.25 μm apart. Raw images were deconvolved using SlideBook.
Liu X., Zhang Z., Ruan J., Pan Y., Magupalli V.G., Wu H, & Lieberman J. (2016). Inflammasome - activated gasdermin D causes pyroptosis by forming membrane pores. Nature, 535(7610), 153-158.
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9

Tracking Airway Dendritic Cells in Mice

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HDM extract (100 µg) was labeled with the Alexa Fluor 647 (AF647) using Protein Labelling Kit (Molecular Probes, Life Technologies) and administered in 50 µL of PBS by i.n. instillation to naïve Balb/c mice 30 minutes after vehicle or DMF treatment (0.5 mg/kg bwt in a total volume of 40 µL). Lungs and mLNs were harvested after 24 hours and the number of Live/SiglecF−/CD11c+/MHCII hi/SSClo/CD11b+/HDM+ DCs were quantified with flow cytometry.
Jaiswal A.K., Sandey M., Suryawanshi A., Cattley R.C, & Mishra A. (2019). Dimethyl fumarate abrogates dust mite‐induced allergic asthma by altering dendritic cell function. Immunity, Inflammation and Disease, 7(3), 201-213.
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10

Fluorescent Protein Uptake Assay

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When used for uptake experiments, Hp, HpSP and BSA were labelled with Alexa Fluor 488
using the protein labelling kit (Life Technologies). The manufacturer's protocol was
adapted, extending the reaction time to overnight at 4 °C to increase labelling
efficiency. Hp and HpSP were then subsequently mixed with Hb to form complex as
above. For each assay, 5 × 106 wild type Lister 427 or
HpHbR−/− cells were resuspended in 100 µl
of serum free HMI-9 with 1% BSA and incubated with 2 µM protease inhibitor
FMK-024 for 10 min at 37 °C. Cells were then incubated with 1–62.5 nM
fluorescently labelled protein for 2 hr at 37 °C before being washed twice in
serum free HMI-9 with 1% BSA. Cells were fixed in 4% paraformaldehyde for 10 min at
room temperature and resupended in PBS. Uptake was assayed by flow cytometry using a
FACScan (BD Biosciences) and quantified on FlowJo software. Mode increase in
fluorescence was measured relative to a no ligand negative control, and all assays
were carried out in triplicate.
Lane-Serff H., MacGregor P., Lowe E.D., Carrington M, & Higgins M.K. (2014). Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor. eLife, 3, e05553.
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