Pd l1 e1l3n xp rabbit mab

Manufactured by Cell Signaling Technology

Sourced in United States

PD-L1 (E1L3N) XP Rabbit mAb is a laboratory reagent that specifically binds to the PD-L1 protein. It is designed for use in various analytical techniques to detect and study the PD-L1 protein.

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PD-L1 (184 citations) E1L3N (126 citations) XP® Rabbit mAb (21 citations) PD-L1 (E1L3N, (19 citations) E1L3N) XP Rabbit mAb (3 citations) PD-L1 (E1L3N) XP (2 citations) PD-L1 mAb (2 citations) Rabbit mABs (2 citations)

5 protocols using pd l1 e1l3n xp rabbit mab

PD-L1 Expression Immunohistochemistry Protocol

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Immunohistochemistry (IHC) was performed on paraffin-embedded specimens fixed in 4 % buffered formalin, using 3‑µm-thick histological sections. For PD-L1 expression in pre- and post-NCR tissue, two different staining methods were used. The first staining method was performed on a Ventana® BenchMark ULTRA (Roche, Tucson, AZ), automated IHC slide staining system, using the universal DAB detection kit with cell conditioning 1 (CC1) buffer of pH 8 and the PD-L1 (E1L3N®) XP® rabbit mAb (Cell Signalling Technology, Danvers, MA).
The second staining method was performed manually, using the antibody 5H1 (monoclonal antibody of human B7H1, Dr. Lieping Chen’s lab, isotype: mouse IGg1) as described previously [18 (link)].
PD-L1 expression was scored as positive or negative with semiquantitative estimation of percentage of positive tumor and stroma cells. Slides stained according to the first staining method were independently reviewed and scored by two observers (M.L. and J.G.). All slides stained according to the second staining method were reviewed and scored by M.B.

Jomrich G., Silberhumer G.R., Marian B., Beer A, & Müllauer L. (2016). Programmed death-ligand 1 expression in rectal cancer. European Surgery, 48(6), 352-356.

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Quantification of Cellular PD-L1 Expression

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Cells were washed twice in phosphate-buffered saline (Beyotime, Shanghai, China), lysed in ice-cold radioimmune precipitation assay (Beyotime) buffer, and then centrifuged for 10 minutes at 4℃. Supernatant was collected, and protein concentrations were determined using the BCA Protein Assays (Invitrogen). Cell lysates were separated by SDS-PAGE (Beyotime) gel and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). Membranes were blotted with 10% nonfat milk, washed in Trisbuffer saline (TBS) with 0.1% Tween (Beyotime) and incubated with primary polyclonal antibodies overnight at 4℃. After washing with TBS-Tween, membranes were incubated with secondary antibody (horseradish peroxidase conjugated IgG; Cell Signaling Technology, Danvers, USA) for 60 minutes at room temperature. Then, they were washed again with TBS-Tween and detected using the ChemiDoc Touch imaging system (Bio-Rad, Carlsbad, USA). PD-L1 (E1L3N®) XP® Rabbit mAb (1:1,000; Cell Signaling Technology) and β-Actin (13E5) Rabbit mAb #4970 (1:2,000; Cell Signaling Technology) were used for Western blotting.

Yang L., Cai Y., Zhang D., Sun J., Xu C., Zhao W., Jiang W, & Pan C. (2018). miR-195/miR-497 Regulate CD274 Expression of Immune Regulatory Ligands in Triple-Negative Breast Cancer. Journal of Breast Cancer, 21(4), 371-381.

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Comprehensive Immunophenotyping of PBL

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The following anti-human monoclonal antibodies (mAb) were used for flow cytometry staining: CD8-FITC, CD137-PE, CD39-PE-Cy7, CD4-AF700, LAG3-PE, PD-1-PE, PD-L1-PE (all eBioscience. Waltham, USA), BLTA-BV421, GITR-BV421, OX40-PE-Cy5, TIM3-PB (all Biolegend, London, UK), and CD45-AMCyan (BD Bioscience, San Jose, CA, USA). All antibodies were pre-titrated using stimulated and non-stimulated PBL to determine optimal staining dilutions. For IHC, PD-L1 (E1L3N) XP Rabbit mAb was purchased from Cell Signaling Technology, Danvers, MA, USA.

Puntigam L.K., Jeske S.S., Götz M., Greiner J., Laban S., Theodoraki M.N., Doescher J., Weissinger S.E., Brunner C., Hoffmann T.K, & Schuler P.J. (2020). Immune Checkpoint Expression on Immune Cells of HNSCC Patients and Modulation by Chemo- and Immunotherapy. International Journal of Molecular Sciences, 21(15), 5181.

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Western Blotting of Cellular Proteins

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For Western blotting, cells were lysed in RIPA buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% NP-40, 2 mmol/L EDTA, 0.5% Na Deoxycholate, and 0.1% SDS] supplemented with protease and phosphatase inhibitor cocktail (Cell Signaling Technology, #5872s). Protein concentrations were measured with BCA Protein Assay Kit (Sangon Biotech, #C503021). For Western blotting, equal amounts of protein were heat denatured in the presence of a reducing agent and separated on 4%–12% or 10% Bis-Tris SurePAGE gels (GeneScript, #M00652, #M00665), and transferred to PVDF membranes. Antibodies used for Western blotting were as follows: anti-mouse MAN2A1 Antibody (Santa Cruz Biotechnology, #sc-376909), anti-human MAN2A1 antibody (Santa Cruz Biotechnology, #sc-377204), p STAT1 (Cell Signaling Technology, #9167), STAT1 (Cell Signaling Technology, #9172), ERK2 (Santa Cruz Biotechnology, #sc-1647), GAPDH (Sigma, #G9545), PD-L1 (E1L3N) XP Rabbit mAb (Cell Signaling Technology, #13684T), vinculin (Santa Cruz Biotechnology, #sc-73614), Goat anti-rabbit secondary antibody (Cell Signaling Technology, #7074s), and Goat anti-mouse secondary antibody (Cell Signaling Technology, #7076s). The blots were imaged using CLINX imaging system according to the manufacturer’s instruction.

Shi S., Gu S., Han T., Zhang W., Huang L., Li Z., Pan D., Fu J., Ge J., Brown M., Zhang P., Jiang P., Wucherpfennig K.W, & Liu X.S. (2020). Inhibition of MAN2A1 Enhances the Immune Response to Anti–PD-L1 in Human Tumors. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(22), 5990-6002.

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PD-L1 Immunohistochemistry Assay Protocols

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All PD-L1 IHC at MSKCC was performed using Cell Signaling Technology’s PD-L1 (E1L3N®) XP® Rabbit mAB (n = 11). Two patients whose PD-L1 testing was performed through an outside institution utilized Dako® PD-L1 IHC 22C3 pharmDx.

Miller K.M., Filippova O.T., Hayes S.A., Abu-Rustum N.R., Aghajanian C., Broach V., Ellenson L.H., Selenica P., Jewell E.L., Kyi C., Lakhman Y., Mueller J.J., O'Cearbhaill R.E., Park K.J., Sonoda Y., Zamarin D., Weigelt B., Leitao MM J.r, & Friedman C.F. (2021). Pattern of disease and response to pembrolizumab in recurrent cervical cancer. Gynecologic Oncology Reports, 37, 100831.

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