Comment[ArrayExpressAccession]	E-GEOD-49605
MAGE-TAB Version	1.1				
Public Release Date	2013-10-01
Investigation Title	Screening for novel immungenic proteins of Salmonella Enteritidis 125109				
Comment[Submitted Name]	Screening for novel immungenic proteins of Salmonella Enteritidis 125109
Experiment Description	The screening of a cDNA derived expression library of Salmonella Enteritidis 125109 expressed in E. coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (fimA) and negative (argC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from S. Enteritidis by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 9 novel immunogenic proteins could be identified. In total 1536 different lysates were spotted on different microarray slides. Each slides contained 3600 distinct spots, seperated into two compartments of 1800 spots each. Each compartment comprised quadruplicate replicates of each sample lysate with controls being analysed with more replicates. As controls we used: fimA from 3 different samples (40 replicates) as positive reference proteins, as it has been described as immunogenic before. GapA from Klebsiella pneumoniae and Campylobacter jejuni (40 replicates) as negative reference proteins, additionally two sets of E.coli cell lysates without fusionprotein expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates). Last but not least a buffer control (24 replicates) was included. Therefore, each set of replicate slides contained 384 different samples. Each screening was performed with three replicate slides. Consequently, a total number of 12 slides were screened (4 sets of samples x 3 replicates each). For identification rabbit polyclonal antibody to S. enterica (Abcam ab35156) as primary and Goat polyclonal to Rabbit IgG conjugated with ChromeoTM-546 (Abcam ab60317) as secondary antibody were used. The top compartment was incubated first with primary antibody, while the bottom compartment was incubated with PBS at the same time. Afterwards both compartments were incubated with secondary antibody.				
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl			
Person Last Name	Hoppe				
Person First Name	Sebastian				
Person Email	sebastian.hoppe@ibmt.fraunhofer.de				
Person Affiliation	Fraunhofer Institute for Biomedical Engineering				
Person Address	Fraunhofer Institute for Biomedical Engineering, Am Mühlenberg 13, Potsdam, Germany				
Person Roles	submitter				
Protocol Name	P-GSE49605-1	P-GSE49605-3	P-GSE49605-4	P-GSE49605-2	P-GSE49605-5
Protocol Description	From the raw data the corrected median fluorescent intensities (F532 median - B532) were used. Then, relative fluorescence intensities were calculated by subtraction the fluorescent intensities of the top compartment by the ones from the bottom compartment. This acknowledges the presence of unspecific interaction of the secondary antibody to the samples. Next, the contrast method was applied by calculating the contrast for each spot of the top compartment. C = [rfi(sample) - median rfi(negative reference)]/[rfi(sample) + median rfi(negative reference)] ID_REF =  VALUE = relative fluorescence intensity	n/a	Fusion proteins were spotted and coupled to the microarray surface for one hour at room temperature with 70% humidity. Slides were washed three times with PBST (PBS + 0.01% Tween20). Incubation with primary antibody (rabbit polyclonal to S. enterica) and secondary antibody (goat polyclonal to rabbit, Chromeo-546 conjugated) were performed for 1.5 h each, with washing (three times) with PBST after each incubation step. Slides were dried with nitrogen and scanned afterwards.	Fusion proteins were extracted from cultures using EasyLyse Bacterial protein extraction kit (Epicentre)	Fluorescent array images were collected for Cy3 with a Genepix 4200A fluorescent scanner. Image intensity data were extracted and analyzed using Origin Pro 8G.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	nucleic acid extraction protocol	array scanning protocol
Comment[SecondaryAccession]	GSE49605				
Comment[GEOReleaseDate]	2013-10-01				
Comment[ArrayExpressSubmissionDate]	2013-08-06				
Comment[GEOLastUpdateDate]	2013-10-02				
Comment[AEExperimentType]	proteomic profiling by array				
SDRF File	E-GEOD-49605.sdrf.txt