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These predicted Human leukocyte antigen [HLA] allele binding peptides were further analyzed for potential conserved region using an Immune Epitope Database and Analysis Resource (IEDB).
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For the thermal stability assay, these cells binding with peptides were further incubated at 37°C for 2 h with or without 1000 ng/mL Brefeldin A (Selleck Chemicals, Burlington, USA).
The remaining potential lead peptides were further filtered by comparing TNF-α SPR binding response to the binding response from three unrelated proteins on the SPR chip as well as the response from the neutravidin coated reference spot.
The binding modes of HLA-B*57 01 HLA-B*57 01ds for all thite peptides were further explored using 3D interacompoundsgerprints and hierarchical clustering.
Selected peptides were further analyzed by MALDI-TOF-TOF MALDI-TOF-TOF MALDI-TOF-TOF MS/MS
The ten A∗0201-binding A∗0201-bindingfurther tested for immune recognition in peptidesal blood mononuclear cells isolated from six A∗0201-positive healthy donors using interferon γ (IFN γ) ELISpot assay.
The binding of transfected cells with peptides was further confirmed by FACS analysis.
The artificial amino acid sequences are then submitted to NetMHC, configured to predict all 55 possible MHC alleles within the software (version 3.0), and only the predicted strongly binding peptides are filtered for further processing.
Evaluation of the best binding peptides was performed via incubation of the peptide array-bounded cells with MTT reagent, which is reduced to purple formazan in living cells and further quantified using an Elispot and Kodak imager.
The 1183 predicted HLA-binding peptides were synthesized and used to generate individual pMHC monomers using UV-mediated peptide exchange32.
For the subset of peptides which were further studied to obtained binding constants, the fluorescent binding assay was completed as described above.
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