ZeptoMARK CeLyA Service Sample Results ZeptoMARK CeLyA Lysate Microarray Experimental Procedure In a ZeptoMARK CeLyA service study, your cell lysates or tissue lysates are spotted on ZeptoMARK chips, after the total protein concentration has been normalized. The ZeptoMARK CeLyA cell lysate arrays are then incubated with your choice of primary antibodies against e.g. signaling proteins, one antibody per array. Utilizing a fluorescence labeled anti-species secondary antibody, the amount of primary antibody bound in each lysate spot in the array is measured in the ZeptoREADER. Duplicate arrays are analyzed for each primary antibody. The ultimate sensitivity of the ZeptoREADER technology enables the researcher to detect minute amounts of bound antibody and thus to quantify relatively low abundant proteins in the lysates. The microarray images are analyzed with the ZeptoView Pro software and the lysate spot intensities are referenced to control spots resulting in Referenced Fluorescence Intensities (RFI). The RFI values for each of your lysates assayed with each primary antibody are the raw data you will obtain from a CeLyA study. Bar plot profiles, EC50 curves and fold change diagrams are simple visualizations of these data. - For details of the experimental procedure please refer to the document “ZeptoMARK CeLyA Process”. Expression and Activation Profiles The measured relative abundance of a protein or a posttranslational protein modification against which the primary antibody is directed is displayed as a bar plot diagram. On the x-axis, 32 lysates spotted in one array are listed. On the y-axis, the RFI value is plotted. The error bars correspond to the variability between the duplicate arrays incubated with the same primary antibody. The example in Figure 1 shows that e.g. in lysate 27 there is 2 times more MAP kinase p44/42 present than in lysate 26. Figure 2 shows the results for the same lysates, this time incubated a primary antibody specifically directed against activated, phosphorylated MAP kinase p44/42. The phosphorylation status of MAP kinase p44/42 in the 32 cell lysates can now be analyzed by comparing Fig. 1 and Fig. 2. For example: Whereas the level of total MAP kinase p44/42 is identical in lysate 6 and 7, the level of activated MAP kinase p44/42 in cell lysate 6 is twice as high as in lysate 7. This indicates an increase in p44/42 MAPK signaling induced by for example the different treatment of the cell cultures 6 and 7. ZeptoMARK CeLyA Service Sample Results EC50 Calculations The efficacy of a drug (EC50 value) can be calculated from results of ZeptoMARK CeLyA arrays representing lysates treated with different concentrations of a compound. In the experiment of Figure 3, cell cultures of 2 different cell lines have been treated with 7 different concentrations of a compound. The cells were lysed, spotted in a ZeptoMARK CeLyA format and the effect of the treatment on the target or other marker protein quantified. Per definition, the EC50 value is the compound concentration where half of the maximum effect is reached. EC50 1.0 0.8 0.6 Fig. 4 Cleaved caspase 3 Cyclin D1 P-Rb P-Histone H3 beta-catenin alpha-catenin P-Stat3 P-SAPK/JNK P-p38 1.2 P-Akt 1.4 P-Erk2 Fold signal changes (treated / control) Biomarker Finding In biomarker finding the relative abundance and activation of proteins is investigated in order to identify markers specific for healthy vs. disease states or treated vs. control samples. Fold changes as displayed in Fig. 4 are a useful way to visualize differences between samples. In this example, different expression and activation levels between colorectal cancer tissue, chemotherapeutically treated vs. control are visualized. Significantly less phosphorylation is observed for Stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). The dashed lines indicate the significance threshold of 20% difference in RFI signal. With the large number of samples that can efficiently be screened with ZeptoMARK CeLyA lysate arrays it becomes feasable to validate biomarkers on a large statistical basis.