Frequently Asked Questions
1. What is the turn-around time for the Kinex™ antibody microarray and Kinetworks™ immunoblotting analyses?
The normal turn-around times for the Kinex™ and Kinetworks™ screening results are three (3) to four (4) weeks from receipt of the samples at Kinexus.
2. What species can be used for Kinex™ and Kinetworks™ analyses?
The antibodies used in the Kinex™ and Kinetworks screens have been optimized to work in human, rat and mouse. We have also used these screens for analysis of other species, with high (eg. guinea pig, hamster, chicken, frog) to reasonable success (eg. sea star, sea urchin). In general, phosphosite antibodies worked over a wide-range of species than pan-specific antibodies.
3. Is the 500 μg quantity necessary for all the target proteins and phosphosites mentioned in the Kinetworks™ screens or is that the amount needed for each target?
A minimum of 300 μg of sample is all that is required to track all the proteins and phosphosites in all of the standard Kinetworks™ KPSS screens. We request extra sample material, because occasionally there can be slight leakage of sample tubes during shipping and inaccuracies in estimated volumes.
4. How do you present the results of the Kinetworks™ analysis?
For each sample you submit for Kinetworks™analysis you will receive the following:
- A TIFF file (or image) of the immunoblot, with the detected bands unmarked.
- A TIFF file (or image) of the immunoblot, with the detected bands numbered.
- An Excel spreadsheet containing quantitative information about band intensity to reference with the immunoblots containing numbered bands with:
- the identities of all the target proteins identified as bands in the Kinetworks™ screen;
- the estimated molecular mass for each numbered band;
- the relative quantity of the band expressed as a percentage of the total intensity of data in the lane in which the band appears;
- the trace quantity of each band taken to be the area under the band's intensity profile curve;
- the corrected trace quantity of each band expressed as counts per minute (cpm);
- for multiple samples, the normalized trace quantity of each band in cpm. This adjustment is based on the difference in the total signal from the sum of the bands on this immunoblot as compared to the average total signal for all of the related immunoblots in a set of multiple samples; and
- a bar graph of target protein changes, including percent change from controls.
- For an additional cost, Kinexus can provide coloured overlays of TIFF images of the immunoblots with target proteins labeled in Powerpoint slides for presentation purposes or various formats (eps, tiff, jpeg) for publication.
5. Can you send an example of how the Kinetworks™ results are presented?
Please see the "Kinetworks™" section for an example of the Kinetworks™ report (www.kinexus.ca/services/kinetworks).
6. Do you have licenses from the original antibody manufacturers to use their products in your screens? (Will this may affect the usability of the data for publication?)
The antibodies that we use in the Kinetworks™ and Kinex™ screens are obtained from over 20 commercial vendors and therefore, licenses from these manufacturers are not required for their usage in our screening service. Therefore, there should also be no problems with regards to using the data in publications.
7. What happens if for some reason the tests don't work? Do I get to send you another batch of protein lysate? Are you going to charge me again?
The methodology used in the Kinetworks™ services has stood up to rigorous testing. To date, Kinexus has performed over 10,000 screens. As proper sample preparation is crucial to obtaining the best results, it is difficult to determine whether problems with the sample or the screening process are the reason why a particular test does not seem to perform as expected. For this reason, Kinexus cannot offer any guarantees on the results obtained with our proteomics analyses. You can be assured that your samples are processed properly at Kinexus as we are just as keen as you are to obtain reliable results.
8. Why is it that the quantitation results might not be the same using different Kinetworks™ screens for the same target proteins?
Different Kinetworks™ screens may include antibodies for the same target proteins from different suppliers. These polyclonal antibodies may have slightly different cross reactivities, for example, against two adjacent phosphorylation sites. These mixtures of antibodies may differ in their relative specificities for the two phosphosites and surrounding amino acid residues.
9. Is it possible to request testing of only one of the protein kinases, but using a number of different samples? For example, would it be possible to provide you with (say) 100 lysates and measure CDK1 Thr-161 phosphorylation only?
This can be done using our Kinetworks™ Custom Services. With KCSS 1.0, you can analyse 8 separate samples with up to three different antibodies (provided that the target proteins have distinct molecular masses) on one immunoblot. Also with KCPS 1.0, you can select 18 antibodies from over 650 to analyse one sample on the same immunoblot.
With our Custom Kinex™ Reverse Lysate Microarray Service, several hundred cell/tissue lysates can be probed at four dilutions, each in triplicate against a desired antibody. However, to use this service, clients must have a minimum of 20 different antibodies tested.
10. Do you reveal or identify which manufacturer's antibodies that you are using for each specific assay so we can verify the results independently?
In optimizing the Kinetworks™ screens, Kinexus has independently tested over 6000 antibodies from over 25 of the leading suppliers of cell signalling protein antibodies, and have incorporated only the best antibodies into the Kinetworks™ screens. This has cost us well over $1 million. Because the vast majority of the antibodies that Kinexus has tested do not work satisfactorily in our screening system, the information about which commercial antibodies perform or failed is an important part of the trade secrets of Kinexus. However, we are willing to reveal the sources of specific antibodies upon request for those proteins that change in a client's samples as revealed by Kinetworks™ analyses. We have provided several hundred client referrals for antibodies over the last 16 years. Over a quarter of the antibodies used in out proteomics services are produced by Kinexus and are now available directly from our company as stand-alone products.
11. I have a lot of samples to analyze but I just can’t afford to test them all with your services. What should I do?
Various strategies can be taken to minimize the number of experiments required and contain cost to obtain the desired results. For example, if you had 4 control samples and 4 treatment samples of lysates from tissues to analyze against 877 antibodies to identify potential biomarkers, we recommend that you pool a portion of the control and treatment samples separately and have a single Kinex™ KAM-880 Antibody Microarray analysis performed. Upon receipt of the Kinex™ results, you should select 18 antibodies that gave strong changes and signal intensities, and have two Kinetworks™ Custom KCPS 1.0 protein screens performed with the separately pooled control and treatment samples. In the selection of these antibodies, a quick query of the on-line KiNET databank may reveal if these antibodies have previously detected the target proteins in these tissues. We expect that at least 6 to 9 of the 18 tested antibodies will confirm the previous changes detected with these antibodies on the Kinex™ antibody microarray. Another different Kinetworks™ Custom KCSS 1.0 multi-sample screen should then be performed for the analysis of the individual control and treatment samples. With this screen, 8 different samples can be analyzed on one immunoblot with up to three different antibodies (provided the targets have different molecular masses) as a single analysis. If you can tell us about the nature of your tissue samples and we can deposit your results in the KiNET databank, then you can save 40%. This strategy will yield highly reproducible results with two alternative methods, and provide information with individual samples to confirm whether the detected target proteins are suitable biomarkers. To validate the identified biomarker candidates against hundreds of clinical samples, we recommend a follow up with our Custom Kinex™ Reverse Lysate Microarray Service.