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Drosophila CG1275 Expression, Antibody Production, and Western Blotting
Author(s) -
Garcia Brandon Michael,
Gorman Maureen J.,
Ragan Emily J.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.795.6
Subject(s) - microbiology and biotechnology , complementary dna , rna interference , gene knockdown , blot , affinity chromatography , cloning (programming) , expression cloning , expression vector , biochemistry , biology , antibody , drosophila melanogaster , chemistry , gene , enzyme , rna , genetics , computer science , programming language , recombinant dna
Little is known about iron absorption in insects. One potential mechanism involves the action of a ferric reductase to reduce Fe(III) to Fe (II), which can enter the cell through a divalent metal transporter. CG1275 is a potential ferric reductase that is found in Drosophila melanogaster. By replicating, cloning, and expressing the N‐terminus of CG1275 we can develop a reagent to allow us to observe a protein knockdown after RNA interference (RNAi) experiments. We cloned a cDNA encoding the N‐terminal 107 amino acids of CG1275‐PA, the region before the first transmembrane helix, into pET‐32a. This was confirmed by restriction digestion. Expression of the N‐terminus of CG1275 along with a thioredoxin (Trx) tag and 6xHis tag from the vector was performed in Bl21 (DE3) E. coli. We then purified the protein using Nickel‐column affinity chromatography. The purified protein was separated by SDS‐PAGE and the protein bands of interest were cut out and sent for antibody production. The newly developed antibodies were then separated to remove antibodies that recognize the Trx tag. The antibodies to CG1275 were then used for western blots of S2 cell lysates to determine if they recognize CG1275 and are able to verify successful CG1275 knockdown after RNAi experiments. Support or Funding Information NSF (IOS‐1656407) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .