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Immunoprecipitation (IP) combined with LC-MS/MS to identify potential plant targets of BcCELP1

ABSTRACT: The raw MS data were analyzed and searched against the target protein database using Maxquant (1.6.2.10). Only peptides identified with high confidence (with enzyme specificity set to trypsin; maximum missed cleavages set to 2; precursor ion mass tolerance set to 20 ppm, and MS/MS tolerance 20 ppm) were chosen for downstream protein identification analysis. To confirm the interaction between BcCELP1 and NbRACK1, a Co-IP assay was conducted as follows: pCNG-BcCELP1, pCNG-EV and pCNF3-NbRACK1 constructs were transiently co-expressed by agroinfiltration of 4-5 week-old N. benthamiana leaves. Total proteins were extracted from leaves using a cell lysis buffer, and subjected to anti-GFP agarose beads IP (chromotek) as previously described45. Eluted proteins were subjected to immunoblot analysis using anti-GFP antibodies and anti-FALG antibodies. Total proteins were loaded as an input control.

ORGANISM(S): Nicotiana Benthamiana

SUBMITTER: Kai Bi

PROVIDER: PXD055429 | iProX | Wed Aug 28 00:00:00 GMT+01:00 2024

REPOSITORIES: iProX

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