´╗┐Researchers can quickly determine presence or absence of specific proteins and examine how each proteins abundance changes in different conditions and strains

´╗┐Researchers can quickly determine presence or absence of specific proteins and examine how each proteins abundance changes in different conditions and strains. Acknowledgments USDA-Agricultural Research Service is an equal opportunity employer. Supplementary Materials Click here for additional data file.(15M, zip) The following are available online at http://www.mdpi.com/2076-2607/6/4/127/s1, Figure S1: mAbs 11F8, 7A6 and 10C12 react to the MAP_3936 coding sequence within phage clone #4-2a, Figure S2: mAb 6C9 binds to MAP_3060c, Figure S3: The antigen that binds 12C9 was captured in a specific manner, Figure S4: Location of antibody epitopes to the MAP_3936 groEL2 protein, Figure S5: MAP_3404 epitope mapping using an overlapping peptide array, Figure S6: Location of the 12C9 antibody epitope to the center region of MAP_4145, Table S1: Recombinant protein assignments on the dot blot array shown in Figure 2B, Table S2: Peptide library sequences and spot/well locations for MAP_2698c and MAP_3404, Table S3: Monoclonal antibody reactivity to mycobacterial species. Author Contributions Conceptualization, J.P.B.; Methodology, J.P.B., J.R.S., J.D.L., and T.A.R.; Investigation, J.P.B., J.R.S., J.D.L., and T.A.R.; Resources, J.P.B., J.R.S., J.D.L., and T.A.R.; Writing-Original Draft Preparation, J.P.B.; Writing-Review and Editing, J.P.B., J.R.S., J.D.L., and CB-6644 T.A.R.; Project Administration, J.P.B.; Funding Acquisition, J.P.B. to remains unknown [2]. The same antigen anonymity is true for mAbs that react to secreted proteins of [3]. CB-6644 To close a research gap and facilitate detection of whole cell extracts or membrane enriched extracts [4,5]. In those studies, only five antigens were successfully identified when screening a phage lambda expression library with the antibodies. The DnaK chaperone (MAP_3840) was identified as the corresponding antigen for mAbs 11G4 and 13A4, along with isocitrate lyase enzyme (MAP_1643) for mAbs 9G10 and 11F6 [4]. A proline-rich antigen (MAP_1025) was later identified using a similar screening approach with mAb 17A12 [5]. However, the remaining five mAbs failed to react with plaques in the phage library and thus their cognate antigens remained unknown. One of these antibodies (4B6) detected a highly conserved protein among all tested mycobacterial species [4]. Because of this lack of specificity, mAb 4B6 was not pursued further. A subsequent study generated 22 additional mAbs in our laboratory that were not published and corresponding antigens were never identified. Those mAbs were examined further in this study. In separate studies, our group also obtained mAbs to select proteins of interest using well-defined recombinant proteins as the immunizing antigen in mice. For example, two mAbs were obtained when immunizing mice with MAP_1272c, a strong antigen that has been shown to hydrolyze peptidoglycan [6,7]. This antigen is a NlpC/P60 domain containing protein that was recently crystalized and shown to have lost the ability to cleave peptidoglycan CB-6644 due to a single amino acid modification in the catalytic triad [7]. The two mAbs successfully developed to this protein each bound distinct epitopes in MAP_1272c [6]. Another two mAbs were obtained when immunizing mice with the 35-kDa major membrane protein [8]. In each study, there was no need to define the cognate antigen, because the recombinant proteins used for immunizing mice were well characterized [7,9]. However, in several other attempts, mAbs were not successfully obtained with recombinant proteins, or more commonly, the resulting Gfap hybridoma secreting antibodies only reacted to the expressed recombinant and not the native protein, highlighting the limitation of this approach. Although we had success identifying cognate antigens by screening a phage expression library of K-10 for several monoclonal antibodies, there were still a number of antibodies that did not show reactivity using this type of screening method. This result was CB-6644 reproducible even after three independent attempts at different times with different personnel. Use of antibodies that bind to unknown antigens in studies can lead to error-prone conclusions. For example, a PD4 mAb was used in cancer research because it specifically bound to tumor cells. The antibody was obtained by immunizing mice with the human gastric cell line MGC803 [10]. However, it was later discovered after a failed cDNA expression library screen that the antigen to PD4 was a membrane protein of that could bind directly to tumor cells [11]. Therefore, when library screening approaches failed, we pursued immunoprecipitation and protein array approaches to identify remaining antigens. We CB-6644 conclude this study by using the newly acquired information to determine relative abundance of selected proteins among the complex (MAC) as an example of how these reagents can quickly interrogate the quality of proteomic preparations. This catalog of monoclonal antibodies should.