Anti ulbp1

Manufactured by R&D Systems

Anti-ULBP1 is a recombinant protein that binds to and neutralizes the ULBP1 protein. ULBP1 is a stress-induced antigen that is recognized by the NKG2D receptor on natural killer cells and certain T cells.

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Lab products found in correlation

Anti ulbp3, by R&D Systems (4 mentions) Anti ulbp2, by R&D Systems (3 mentions) Anti mica, by R&D Systems (2 mentions) Anti ulbp 2 5 6, by R&D Systems (2 mentions) Anti micb, by R&D Systems (2 mentions) Anti cd56, by BD (1 mentions) Anti nkg2d antibody, by R&D Systems (1 mentions) Anti cd107a lamp 1, by BioLegend (1 mentions) Anti cd3, by BioLegend (1 mentions) Pomalidomide, by Merck Group (1 mentions) Fitc mouse igg1, by BioLegend (1 mentions) Anti nkg2d, by R&D Systems (1 mentions) Lenalidomide, by Abcam (1 mentions) Phthalimide, by Merck Group (1 mentions) Anti cd107a fitc h4a3, by BD (1 mentions) Anti cd138 fitc m15, by BD (1 mentions) Anti cd38 apc hit2, by BD (1 mentions) Anti mica b, by R&D Systems (1 mentions) Anti rabbit hrp, by Jackson ImmunoResearch (1 mentions) Anti rabbit biotin, by Jackson ImmunoResearch (1 mentions)

5 protocols using anti ulbp1

Quantification of ULBP Proteins by ELISA and Flow Cytometry

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Anti-ULBP1, anti-ULBP2 and anti-ULBP3 antibodies were purchased from R&D systems (catalog numbers: MAB1380, 1248 and 1517 respectively) and used both for flow cytometry and for ELISA assays. Anti-ULBP1 antibody, (catalog number Sc33564, Santa Cruz Biotechnology) was used for western blotting of ULBP1. The W6/32 mAb was used for MHC-I staining. Anti-NKG2D antibody was purchased from R&D Systems (MAB139). The anti-CD99 (12E7) was used as an isotype control. Anti-CD107a (LAMP-1) was purchased from BioLegend (catalog number 328620). Anti-CD56 (Becton Dickinson) and anti-CD3 (BioLegend) antibodies were used to determine NK purity. Anti-VP2/3 and agnoprotein antibodies were produced in house as well as the rabbit polyclonal antibodies against VP1. Anti-T-Ag antibody was purchased from Abcam (Pab416). The commercial recombinant ULBP-1 Fc chimeric protein (R&D systems, catalog number 1380-UL) was used for the generation of ULBP1 standard curve.
CD16-Ig, NKp30-Ig and NKp46-Ig and NKG2D-Ig fusion proteins were generated in the human embryonic kidney 293T cells and were purified on a protein G column as described [38 (link)]. The fusion proteins used in this work were regularly assayed by SDS-PAGE protein gels, to ensure that the proteins were not degraded. Protein purity of all Ig fusion proteins used in this study was approximately 100%.

Bauman Y., Drayman N., Ben-Nun-Shaul O., Vitenstein A., Yamin R., Ophir Y., Oppenheim A, & Mandelboim O. (2016). Downregulation of the stress-induced ligand ULBP1 following SV40 infection confers viral evasion from NK cell cytotoxicity. Oncotarget, 7(13), 15369-15381.

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Immunophenotyping of Tumor Cells and Natural Killer Cells

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Lenalidomide was purchased from (BioVision Inc.), Pomalidomide and Phthalimide were purchased from (Sigma-Aldrich). These drugs were dissolved in dimethylsulphoxide (DMSO) and stored at −20°C until use. The final concentration of DMSO in all experiments was < 0.1%.
The following monoclonal antibodies (mAbs) were used for immunostaining or as blocking Abs: anti-MICA (MAB159227), anti-MICB (MAB236511), anti-ULBP-1 (MAB170818), anti-ULBP-2/5/6 (MAB165903), anti-ULBP-3 (MAB166510) and anti-NKG2D (MAB149810) from (R&D System), anti-PVR/CD155 (SKII.4) kindly provided by Prof. M. Colonna (Washington University, St Louis, MO), anti-CD56 (C218) mAb was provided by Dr. A. Moretta (University of Genoa, Genoa, Italy), anti-DNAM-1 (DX11) from (Serotec), APC Goat anti-mouse IgG (Poly4053), anti-CD3/APC (HIT3a), anti-CD56/PE (HCD56), mouse IgG1/FITC, /PE or /APC isotype control (MOPC-21) were purchased from (BioLegend). Anti-CD107a/FITC (H4A3), anti-CD138-FITC (M15) and anti-CD38-APC (HIT2) were purchased from (BD Biosciences).

Fionda C., Abruzzese M.P., Zingoni A., Cecere F., Vulpis E., Peruzzi G., Soriani A., Molfetta R., Paolini R., Ricciardi M.R., Petrucci M.T., Santoni A, & Cippitelli M. (2015). The IMiDs targets IKZF-1/3 and IRF4 as novel negative regulators of NK cell-activating ligands expression in multiple myeloma. Oncotarget, 6(27), 23609-23630.

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Evaluation of Tumor-derived Soluble NKG2DL Inhibition

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T cells were resuspended in complete medium with a density of 1×10⁶ cells/ml and were cultured with anti-CD28 (2 µg/ml) and IL-2 (10 U/ml) in a 24-well plate pre-coated with 5 µg/ml anti-CD3. Subsequently, T cells were co-cultured with or without 100 µl serial diluted tumor sup for 24 h. For neutralizing sNKG2DL, single and combined soluble NKG2DL neutralizing antibodies (1, 2, or 5 µg/ml anti-MICA/B, ULBP1, ULBP2, and/or ULBP3) were added to T cells and to the tumor cell supernatant co-culture system. Then, these T cells were harvested and prepared for flow cytometric analysis. Neutralized antibodies (anti-MICA/B, anti-ULBP1, anti-ULBP2, and anti-ULBP3) were purchased from R&D Systems, Inc. (cat. nos. MAB13001, MAB1380, MAB1298 and MAB1517, respectively).

Zhang Y., Luo F, & Dong K. (2023). Soluble NKG2D ligands impair CD8+ T cell antitumor function dependent of NKG2D downregulation in neuroblastoma. Oncology Letters, 26(1), 297.

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Comprehensive Antibody Panel for Immunoanalysis

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The following primary antibodies were used for flow cytometry: anti-MICA (clone 159227; R&D Systems), anti-MICB (clone 236511, R&D Systems), anti-ULBP1 (clone 170818; R&D Systems), anti-ULBP2/5/6 (clone 165903; R&D Systems) and anti-ULBP3 (clone 166514, R&D Systems).
The following primary antibodies were used for immunofluorescence: anti-PDI (ab3672, Abcam), anti-FLAG tag (Clone L5, Biolegend) and anti-MICA (clone 159227, R&D Systems).
The following primary antibodies were used for western blotting: anti-MICA (Clone EPR6568, Abcam), anti-FLAG tag (Clone L5, Biolegend), anti-GAPDH (clone 6C5, Santa Cruz) and anti-vinculin (clone EPR8185, Abcam).
The following secondary antibodies were used: anti-mouse AlexaFluor 647, anti-mouse PE, anti-mouse biotin, anti-rabbit biotin, anti-rat biotin, anti-rabbit Cy3, anti-rat 488, streptavidin-AlexaFluor 647, streptavidin-horseradish peroxidase (HRP), anti-mouse-HRP, anti-rat-HRP and anti-rabbit-HRP, all purchased from Jackson Laboratories.

Seidel E., Dassa L., Schuler C., Oiknine-Djian E., Wolf D.G., Le-Trilling V.T, & Mandelboim O. (2021). The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing. PLoS Pathogens, 17(5), e1008807.

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Flow Cytometric Evaluation of Stress-Induced Ligands

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Cells were stained with a mouse isotype IgG2b antibody (Biolegend), with anti-MICA, anti-MICB, anti-ULBP1, anti-ULBP2 (the antibody also recognizes ULBP5 and ULBP6), anti-ULBP3 antibodies (all obtained from R&D Systems), or with the anti-HLA-A, B, C monoclonal antibody W6/32 antibody (purified from the hybridoma). 0.2 µg primary antibodies per staining were added in 100 µL PBS (2 µg/mL) supplemented with 1% BSA and 1mM EDTA and incubated for about 30 minutes at 4°C. Following one wash, the primary antibody was detected using an anti-mouse IgG antibody coupled to Alexa Fluor 647 (Jackson ImmunoResearch) for another 30 minutes at 4°C. For the characterization of cells overexpressing viral genes, staining of the stress-induced ligands was assessed on GFP positive cells.

Chaouat A.E., Seliger B., Mandelboim O, & Schmiedel D. (2021). The HHV-6A Proteins U20 and U21 Target NKG2D Ligands to Escape Immune Recognition. Frontiers in Immunology, 12, 714799.

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