Comparison of proteins extracted from four FFPE tissues and the corresponding cryo tissues with EXB Plus.

<p><b>A</b> Cryo tissue was additionally extracted with EXB Plus but without any heat and T-Per, a buffer designed for extraction of cryo material. To be able to compare protein yields from different sized tissue pieces the protein amount is calculated per mm³ of used tissue. The yields extracted from cryo material with EXB Plus or T-Per were equal, or especially for stomach tissue even higher in the EXB Plus extracts. Cryo tissue compared to FFPE material discloses a loss of protein amount in two tissues (colon and muscle) and no difference in the two others (ovarian carcinoma and stomach). <b>B</b> Western blot analysis of protein lysates from colon FFPE and cryo material. For both tissue types we obtained clear bands in the western blot, confirming that we were able to extract non-degraded, full-length, and immunoreactive proteins. In addition it is visible that the extracted protein amount from FFPE is higher than from fresh-frozen tissue, but hardly any difference could be seen for cryo material extracted using EXB Plus compared to a standard buffer for cryo extraction (T-Per).</p

Comparison of proteins extracted from five different tissues with EXB and EXB Plus, respectively.

<p><b>A</b> Using EXB Plus considerably higher lysate concentrations could be obtained for each of the tissue types. <b>B</b> Western blot analysis of E-Cadherin, Erk and β-actin in five different tissues. For both buffers we obtained clear bands in the western blot, confirming that we were able to extract non-degraded, full-length, and immunoreactive proteins. In addition it is distinguishable that the extracted protein amount from all five tissues of all three analysed proteins is higher using EXB Plus.</p

Protein amounts and western blot analysis of lysates extracted from lymph node samples fixed for 6 h, 24 h, 48 h and 144 h, respectively.

<p>For a short fixation time no real difference could be detected between the two buffers tested. But already at the standard fixation time of 24 h EXB Plus shows 25% higher protein yield. For a fixation time of 144 h the difference increased to 30% higher protein yield compared to EXB. <b>B</b> Western blot analysis of Hsp70, Akt, Erk and β-actin in lymph node samples fixed for 6 h, 24 h, 48 h or 144 h. Protein yield decreases with extension of fixation for EXB, for all four analysed proteins. In contrast, using EXB Plus the decline is much less pronounced. All eight samples detected with one antibody were run on one gel and detected under exactly the same conditions (e.g. blocking, washing steps, exposure time).</p

Protein extraction from FFPE samples from two different xenograft mouse models.

<p>For both mouse models EXB Plus resulted in higher protein yields compared to EXB. This shows the advantage of EXB Plus independently of the organism.</p

Protein amounts gained by extraction from FFPE tissues from two different hospitals in three technical replicates.

<p>EXB Plus allowed high protein yields independently of the tumor sample or the hospital the tissue was processed in. Additionally only minimal variations between the replicates (mean standard deviation: 0.47 mg/ml) could be detected.</p

Protein yield from long-term stored FFPE tissue blocks.

<p>Protein yields from one pair of colon carcinoma and two pairs of gastric cancer tissues from 1990 and 2010 were analysed. The same block was extracted twice, once with EXB and once with EXB Plus. The protein amounts from the newer blocks were higher than from the older ones. But for both time periods the amount extracted with EXB Plus was higher.</p

Protein extraction from FFPE samples from 1990 in comparison to samples from 2010.

<p>For both years the usage of EXB Plus resulted in higher protein yields compared to EXB but for the twenty years old samples the difference was more striking.</p

Impact of sampling method on identification of proteins differentially expressed in tumors vs. normal tissues.

*<p>randomly selected single tumor samples per case; this analysis was repeated 3 times (tumor samples 1–3).</p><p>+ and − symbols after p-values indicate significant upregulation (+) or downregulation (−) of the respective protein in tumor vs. normal tissues.</p><p>n.s., not significant (>0.05).</p><p><i>Italics</i> indicate the group of proteins identified as differentially expressed by all four approaches.</p

Comparison of intratumoral heterogeneity and variation among patients for all proteins, phosphorylated and non-phosphorylated proteins.

<p>CV, coefficient of variation.</p><p>Overall, all proteins combined; Phospho, all phosphorylated proteins combined; Non-phospho, all non-phosphorylated proteins combined.</p><p>Healthy tubes, fallopian tube epithelium from healthy individuals.</p><p>Contralateral tubes, morphologically normal contralateral fallopian tube epithelium from ovarian cancer patients.</p

Variation between 88 individual tumor samples from 13 patients assessed by non-supervised hierarchical clustering based on expression of 36 proteins.

<p>Different patients are color-coded as indicated in the figure legend. High relative expression of proteins is shown in red and low expression in green color. Grey spaces indicate missing data points.</p