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Biotinylated peptide

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Biotinylated peptides are synthetic molecules composed of amino acid sequences with a biotin moiety attached. Biotin is a small molecule with high-affinity binding to streptavidin or avidin proteins, enabling various biotechnological applications.

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Lab products found in correlation

Protease inhibitor cocktail, by Abcam (2 mentions) D1 d2 500ng, by Merck Group (2 mentions) D1 500ng, by Abcam (2 mentions) Pierce pull down biotinylated protein protein interaction kit, by Thermo Fisher Scientific (2 mentions) Pe labelled fluorescent streptavidin, by BioLegend (2 mentions) Cd45ro, by BioLegend (1 mentions) Streptavidin conjugated magnetic beads, by Thermo Fisher Scientific (1 mentions) Scaffold software, by Proteome Software (1 mentions)

11 protocols using biotinylated peptide

1

Identifying DNA-Binding B Cells in SLE Patients

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The DWEYSVWLSN peptide can behave as a dsDNA mimetope [26 (link)]. Immunization of BALB/c mice with an octameric form of this peptide results in the production of anti-dsDNA antibodies, which are involved in lupus nephritis by deposition in renal glomeruli [27 (link)–29 (link)]. Here, a DWEYSVWLSN peptide was used to identify DNA tetramer-binding B cells in SLE patients. A biotinylated peptide was synthesized (GenScript, NJ, USA) and used in tetramerization by combining 5 μl of biotinylated peptide stepwise in 1/2 volumes to 3 mM SA-R-phycoerythrin-PE at a molar ratio of 10:1 and incubating for 60 min at 4 °C.
Wangriatisak K., Thanadetsuntorn C., Krittayapoositpot T., Leepiyasakulchai C., Suangtamai T., Ngamjanyaporn P., Khowawisetsut L., Khaenam P., Setthaudom C., Pisitkun P, & Chootong P. (2021). The expansion of activated naive DNA autoreactive B cells and its association with disease activity in systemic lupus erythematosus patients. Arthritis Research & Therapy, 23, 179.
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2

Identification of Lupus-Associated B Cells

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The DWEYSVWLSN peptide can behave as a dsDNA mimetope [26] . Immunization of BALB/c mice with an octameric form of this peptide results in the production of anti-dsDNA antibodies, which are involved in lupus nephritis by deposition in renal glomeruli [27] [28] [29] . Here, a DWEYSVWLSN peptide was used to identify DNA tetramer-binding B cells in SLE patients. A biotinylated peptide was synthesized (GenScript, NJ, USA) and used in tetramerization by combining 5 µl of biotinylated peptide stepwise in 1/2 volumes to 3mM SA-R-phycoerythrin-PE at a molar ratio of 10:1 and incubating for 60 min at 4 o C.
Wangriatisak K., Thanadetsuntorn C., Krittayapoositpot T., Leepiyasakulchai C., Suangtamai T., Ngamjanyaporn P., Khowawisetsut L., Khaenam P., Setthaudom C., Pisitkun P., & Chootong P. (2021). The expansion of activated naive DNA autoreactive B Cells and its association with disease activity in Systemic Lupus Erythematosus patients.
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3

Detecting αvβ6 and CAR Expression

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Expression of αvβ6 was detected using the 6.3G9
antibody,46 (link) followed by goat
anti-mouse Ig-PE (Dako, Ely, UK) and was expressed as percentage positivity and/
or geometric mean fluorescence intensity. Cells stained with secondary antibody
only served as a negative control. Expression of CARs was detected using
supernatant derived from the 9e10 hybridoma (ECACC), which binds to residues
410-419 of human c-myc, followed by goat anti-mouse Ig-PE. Untransduced T-cells
acted as a negative control. CD8 expression was detected using PE-conjugated
PNIM0452 (Immunotech, Marseille, France). Phenotypic analysis of T-cells was
performed using anti-CCR7 (R&D systems FAB197F) and CD45RO (Biolegend
304210) antibodies. In some assays, populations were gated on CAR+cells detected using an antibody against CD124 (BD Pharmingen, 552178). To
assess integrin specificity of the A20 peptide, biotinylated peptide (Genscript,
Hong Kong Ltd) was diluted in PBS supplemented with MgCl2 and NaCl
(Sigma D8662) and incubated with cells on ice for 20 minutes before being
stained with streptavadin-PE (LifeTech S866) for a further 20 minutes on ice.
Flow cytometry was performed using a FACSCalibur cytometer with CellQuest Pro
software or Fortessa cytometer with FACSDiva software.
Whilding L.M., Parente-Pereira A.C., Zabinski T., Davies D.M., Petrovic R.M., Kao Y.V., Saxena S.A., Romain A., Costa-Guerra J.A., Violette S., Itamochi H., Ghaem-Maghami S., Vallath S., Marshall J.F, & Maher J. (2017). Targeting of Aberrant αvβ6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells. Molecular Therapy, 25(1), 259-273.
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4

Affinity Purification of PTPσ Interactors

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For pulldown experiments, we used the Pierce Pull-Down Biotinylated Protein:Protein Interaction Kit (Thermo Scientific 21115). 100µg/ml of biotinylated-peptide (Genscript) was incubated overnight on an orbital shaker at 4°C with streptavidin beads. Following incubation, extra biotin was added and allowed to incubate overnight to ensure the binding of all streptavidin. After three washes with TBS, either recombinant GST tagged-PTPσ ICD (D1/D2-500ng, Sigma, D1 500ng, Abcam), spinal cord lysate from either wild type or PTPσ null mice, or brain lysate from an adult female Sprague Dawley rat. Neural tissue was quickly extracted and flash frozen with liquid nitrogen. The tissue was homogenized in Tissue Homogenization Buffer (20mM Tris, 0.5mM EDTA, 0.5mM EGTA and 8% Sucrose, pH 7.4) and 1:500 Protease inhibitor cocktail (Abcam) on ice. The lysate was centrifuged at 13,000 rpm for 20 min before addition to the beads. 150µl of each lysate was added to the beads and allowed to incubate overnight at 4°C. Following three washes, beads were incubated with elution buffer for 10 minutes at room temperature. Beads were then centrifuged at 12,000 rpm to collect eluted lysate.
Lang B., Cregg J., DePaul M., Tran A., Xu K., Dyck S., Madalena K., Brown B., Weng Y., Li S., Karimi-Abdolrezaee S., Busch S., Shen Y, & Silver J. (2014). Modulation of the proteoglycan receptor PTPσ promotes recovery after spinal cord injury. Nature, 518(7539), 404-408.
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5

Affinity Purification of PTPσ Interactors

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For pulldown experiments, we used the Pierce Pull-Down Biotinylated Protein:Protein Interaction Kit (Thermo Scientific 21115). 100µg/ml of biotinylated-peptide (Genscript) was incubated overnight on an orbital shaker at 4°C with streptavidin beads. Following incubation, extra biotin was added and allowed to incubate overnight to ensure the binding of all streptavidin. After three washes with TBS, either recombinant GST tagged-PTPσ ICD (D1/D2-500ng, Sigma, D1 500ng, Abcam), spinal cord lysate from either wild type or PTPσ null mice, or brain lysate from an adult female Sprague Dawley rat. Neural tissue was quickly extracted and flash frozen with liquid nitrogen. The tissue was homogenized in Tissue Homogenization Buffer (20mM Tris, 0.5mM EDTA, 0.5mM EGTA and 8% Sucrose, pH 7.4) and 1:500 Protease inhibitor cocktail (Abcam) on ice. The lysate was centrifuged at 13,000 rpm for 20 min before addition to the beads. 150µl of each lysate was added to the beads and allowed to incubate overnight at 4°C. Following three washes, beads were incubated with elution buffer for 10 minutes at room temperature. Beads were then centrifuged at 12,000 rpm to collect eluted lysate.
Lang B., Cregg J., DePaul M., Tran A., Xu K., Dyck S., Madalena K., Brown B., Weng Y., Li S., Karimi-Abdolrezaee S., Busch S., Shen Y, & Silver J. (2014). Modulation of the proteoglycan receptor PTPσ promotes recovery after spinal cord injury. Nature, 518(7539), 404-408.
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6

Pull-down analysis of Dectin-1 and CD69 interactions

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For pull-down assays, C57BL/6 mice were subcutaneously injected with Flt3 ligand-expressing B16 melanoma cells in order to expand the pool of dendritic cells (DC) in the spleen (27 (link)). After 12 days, CD11c+ cells were isolated and total lysates were prepared in lysis buffer (TBS - 1% NP40). Soluble lysates or, alternatively, recombinant tSHIP-1 (28 (link)) were incubated with biotinylated peptides (GenScript, Piscataway, NJ) corresponding to the cytoplasmic tail of mouse Dectin-1 (NH2-MKYHSHIENLDEDGYTQLDFSTQDIHKR-C, biotin in C-terminal lysine), phosphorylated or not in the Tyr3 and 15, or mouse CD69 (NH2-MDSENCSITENSSSHLERGQKDHGTSIHFEK-C, biotin in C-terminal lysine). Peptides were pulled down using streptavidin-conjugated magnetic beads (Life Technologies, Carlsbad, CA) and interacting proteins were analyzed by mass spectrometry or western blotting using antibodies against SYK (N-1), SHIP-1 (P1C1) for Fig. 1C and Supplemental Fig. 1B or (D20 and V19) for Fig. 1D (Santa Cruz, Dallas, TX), LYN (C13F9) and SHP-2 (#3752) (Cell Signaling). Scaffold software (Proteome software, Runcorn, UK) was used to analyze mass spectrometry data.
Blanco-Menéndez N., del Fresno C., Fernandes S., Calvo E., Conde-Garrosa R., Kerr W.G, & Sancho D. (2015). SHIP-1 couples to the Dectin-1 hemITAM and selectively modulates reactive oxygen species production in dendritic cells in response to C. albicans. Journal of immunology (Baltimore, Md. : 1950), 195(9), 4466-4478.
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7

Peptide Pulldown for CaMKK2 Binding

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Peptide pulldown was performed using the PierceTM Biotinylated Protein Interaction Kit (Thermo Scientific) according to the manufacturers’ instructions with LNCaP cell lysate grown in steady-state conditions or following 24 h 10 nM R1881 treatment or ethanol control. Two biotinylated peptides (GenScript, NJ, USA) were synthesised, the Gemin4 CaMKK2 Binding Motif (SLTSFSQNA) and a control peptide (LQPHPVTPS).
Stewart L.M., Gerner L., Rettel M., Stein F., Burrows J.F., Mills I.G, & Evergren E. (2021). CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation. Cell Death & Disease, 12(11), 1040.
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8

In Vitro Protein Synthesis Assay

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All oligonucleotides were obtained from integrated DNA technologies. The biotinylated peptides used for all bead assays were obtained from Genscript (Piscataway, NJ). The FRET peptide used in the progress curves was obtained from Anaspec, and the peptides used for the kinetic assays were synthesized in house on an ABI 433A peptide synthesizer. Streptavidin coated polystyrene microbeads were obtained from Bangs Laboratories. Transcription and translation reagents were made based on (Liu et al. 2005 (link)). A complete list of primer sequences can be found in the Supplemental Methods.
Gianella P., Snapp E, & Levy M. (2016). An in vitro compartmentalization based method for the selection of bond-forming enzymes from large libraries. Biotechnology and bioengineering, 113(8), 1647-1657.
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9

Identifying Targetable Epitopes Using Bioinformatics and Tetramerization

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In order to determine which peptide would be best targeted by an
antibody, the amino acid sequence for SDCBP was run through two epitope
prediction tools (Bepipred Linear Epitope Prediction 2.0 and ABCPred) to
determine predicted epitopes. Predicted targetable extracellular domains were
chosen as the peptides for tetramer analysis. We tetramerized two biotinylated
peptides (GenScript) contained in the extracellular domain of syndecan binding
protein (SDCBP) using PE-labelled fluorescent streptavidin (BioLegend, 405203)
and used flow cytometry to determine the percent of specific antigen-reactive
immortalized B cells which are secreting antibodies against the target molecule
from each of the six immortalized B cell lines, using the protocol previously
described.5 (link) We then
used fluorescence-activated cell sorting with EEEIRANVAVVSGAPL peptide to sort
endometrioid carcinoma-derived immortalized B cells specific for SDCBP from the
pool of immortalized B cells.
Handley K.F., Mehta S., Martin A.L., Biswas S., Maharaj K., Nagy M.Z., Mine J.A., Cortina C., Yu X., Sprenger K., Mandal G., Innamarato P., Powers J.J., Harro C.M., Chaurio R.A., Anadon C.M., Shahzad M.M., Flores I, & Conejo-Garcia J.R. (2023). Actionable spontaneous antibody responses antagonize malignant progression in ovarian carcinoma. Gynecologic oncology, 173, 114-121.
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10

Quantifying MT1-MMP Cytosolic Tail Interactions

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Streptavidin binding plates (96 wells, Pierce) were incubated at 4 °C for 3 h in 0.1 M Na2CO3 buffer (pH 9.6) containing 40 µM of biotinylated peptides (GenScript, Piscataway, NJ, USA) corresponding to the MT1-MMP cytosolic tails: MT1-MMP (563RRHGTPRRLLYCQRSLLDKV582), MT1MMP-RRH563AAA, MT1-MMP-RR569AA and MT1-MMP-R576A. To avoid non-specific binding to biotinylated peptides, samples were blocked with 2% BSA/TBS-Tween containing 10% FBS overnight at 4 °C. GST-tagged recombinant N-moesin [60 (link)] was added to the wells in equal concentrations and incubated at room temperature (RT) for 1 h. After extensive washing, GST protein binding to the cytosolic tail sequences was detected using anti-GST ab and HRP-based detection. Optical density of an empty well was subtracted from all the absorbance data obtained.
Suárez H., López-Martín S., Toribio V., Zamai M., Hernández-Riquer M.V., Genís L., Arroyo A.G, & Yáñez-Mó M. (2020). Regulation of MT1-MMP Activity through Its Association with ERMs. Cells, 9(2), 348.
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