ProteoScan Cancer Lysate Array FAQs

1. What is the ProteoScan Cancer Lysate Array?

The ProteoScan Cancer Lysate Array is an array of 432 protein extracts from cancer and normal tissues, normalized by total protein concentration and spotted onto Nitro-cellulose slides.

2. How many samples and cancer types are represented on the array?

There are 432 samples from 11 types of tissue. Each tissue is represented by 40 samples; 25 samples are from cancer tissues and 15 are from normal tissues. Some tissues have fewer samples due to limited availability (Prostate 13 normal; Melanoma 22 cancer and 14 normal; Liver 23 cancer). There are four different protein concentrations for each sample and these are spotted in triplicate.

3. How were these tissue samples obtained?

Biospecimens were obtained from accredited academic medical institutions in the United States. Samples and data were collected under the highest bioethical principles using IRB and full adherence to HIPAA guidelines.

4. What clinical data accompany each sample?

Samples come with detailed pathology reports AJCC TNM staging and grading; diagnostic tests performed at the medical center; and sample composition (%tumor, %normal, %stroma, %necrosis) as recorded by a board-certified pathologist while reviewing an H&E slide of the sample. Data is coded to protect donor identities.

5. I purchased both the ProteoScan Cancer Lysate Array and the ProteoScan Assay Validation Array. How can I distinguish between the two?

There are several ways to determine which slide belongs to which array: First, the protective container contains only slides from one type of array and it is clearly labeled. Second, The ProteoScan Assay Validation Array is specifically labeled with the product SKU. Third, the nitrocellulose dimensions of the validation slide are smaller (51mm length) that those of the ProteoScan Cancer Lysate slide (60 mm). Fourth, Scan for Cy3 or Cy5 and determine the slide layout based on the BSA-cy3 or BSA-cy5 number and location. If you are still unable to distinguish between them call OriGene technical support for help.

6. Was laser capture micro-dissection or any other micro-dissection procedure performed to separate tumor cells from non-tumor cells?

No, the tissue was not micro-dissected to separate tumor from non-tumor cells. The selected samples have high tumor content as determined by OriGene's pathologist. The specific composition of each sample is available on the datasheet from OriGene's website.

7. Were any preservatives or embedding agents used?

The tissue samples are frozen in OCT, which serves as an excellent protectant against the effects of long term '80-|C storage. The OCT is removed prior to processing the tissue for protein extraction.

8. Are the proteins in the array denatured?

The extraction is performed without denaturating agents such as SDS, urea etc. We used only mild non-denaturating detergents (NP40 and DOC) to preserve the native structure of soluble proteins as much as possible.

9. Why do you add PhosStop to the extracted samples?

The phosphorylation state of many proteins is significantly altered in many cancers. The addition of PhosStop phosphatase inhibitor cocktail preserves these important modifications for detection using phospho-specific antibodies.

10. What changes can be detected using the ProteoScan Cancer Lysate Array?

The array can be used to detect quantitative changes in protein expression profiles, post-translational modifications such as phosphorylation and other changes provided you have a suitable specific antibody.

11. Why are there four different protein concentrations on the array?

The spotted sample includes abundant and less-abundant proteins. To obtain quantitative or semi-quantitative data it is important to cover a large dynamic range. Often, the slope of the Ab response over the 4 dilutions can be correlated with the amount of that particular protein.

12. Were any of the normal and cancer samples taken from the same donors?

Some samples are paired but the majority are not. These paired samples can be identified by the identical pathology report numbers listed for different samples.

13. What methods of detection can we use?

In principal you can use colorimetric, fluorescence and chemiluminescent methods for detection. Certain methods may require the use of special equipment (reader) and the development of an enhancement protocol to increase sensitivity. We routinely use enhanced (TSA) fluorescence and colorimetric methods for detection.

14. Can we purchase individual protein lysates from specific samples included in the array?

Yes, we maintain a stock of protein lysates for most samples. You can purchase these samples directly from OriGene by giving the case number.

15. How can we normalize the results to the relative amount of cell extract spotted?

There is no perfect solution for this problem. We pre-normalized the array based on total protein in each sample. You can use the expression results from one or more housekeeping proteins such as +| -actin, GAPDH, +| and +| tubulin etc. However, the expression of these genes can vary among different tissues and also display differences between tumor and normal tissues. In fact, we detected such differences using +| -actin as a probe. These possibilities should be taken into account when analyzing the data

16. Why should I use the ProteoScan Assay Validation Array?

Before testing the ProteoScan Cancer Lysate Array it is vital to validate the primary and secondary antibodies, identify the appropriate usage dilution and optimize the experimental procedure. The primary difference between the Assay Validation array and the ProteoScan Cancer Lysate array is sample identity-all other components of the two arrays are identical. Thus, this is the most cost-effective method for testing conditions and reagents that will be used with the ProteoScan Cancer Lysate array.

17. Why are the Assay validation slides not included with the ProteoScan Cancer Lysate array?

We prefer to give our customers the freedom to choose according to their needs. While we believe that using our validation array for optimization is the best option not all scientists will require this step. By providing the ProteoScan Cancer Lysate and ProteoScan Assay Validation arrays as separate items, OriGene is able to minimize the cost of the product for all customers.

18. Why is the identity of the samples in the Assay Validation Array not disclosed?

The ProteoScan Assay Validation Array is a supporting tool to help customers achieve success with the ProteoScan Cancer Lysate Array. Many of the samples are a mixture of several lysates from the same tissue type and thus are not representative of a specific disease stage. Because the samples are pooled, it is not possible to accurately link them to clinical data as with the ProteoScan Cancer Lysate array

19. I have an Ab that is shown by the manufacturer to be positive by Western blot. Is it necessary to test it with the ProteoScan Assay Validation Array?

We highly recommend that you do. We have tested several antibodies shown by the provider to be highly reactive by Western blot that completely failed to give any response with the array or even with lysate from cell lines. Using the ProteoScan Assay Validation Array can save you the expense, frustration and time associated with such reagents.

20. My data indicate a high level of background. What can I do to reduce this?

There are several possible causes. The most likely cause of high background is using too high of a concentration of antibody for probing. We suggest diluting the primary antibody and screening with OriGene Assay Validation Array to determine the optimal antibody dilution factor. Higher background can also be attributed to secondary antibodies. Make sure you use qualified secondary antibodies and use appropriate dilution. If TSA enhancement with Biotin-tyramide was used, we recommend extending the blocking time or changing the blocking buffer so that the signal from endogenous biotin is minimized. High background can also come from endogenous peroxidase. Quenching endogenous peroxidase can be achieved by treating the sample with 3% H2O2 in PBS for 10 minutes.

21. I didnG??t get any signal from any of the samples or the controls.

The IgG control will almost always give a positive signal if the procedure was executed properly. Most likely, an essential component was not added or a step was missed.

22. The signal from the experimental samples is very low but the signal from the controls is very good.

It is likely that the primary antibody is not sufficiently sensitive for use on the array or that the correct dilution factor was not used. We suggest further testing the primary antibody by screening with OriGene Assay Validation ArrayG?¥ to determine the optimal antibody dilution factor

23. After developing the array, a streak that looks like a comet has appeared.

Unless this streak covers a spot, it will not affect the reading of the actual data from the array. If it does cross a spot, it will be necessary to remove that data point from the analyses. There are two possible causes for this; one, the array became dehydrated at some step during the screening process. It is essential to keep the array hydrated until the final drying step. Alternatively, the solutions used for screening may not have been thoroughly mixed before they were added to the array.