Frequently Asked Questions
What is LC/MS/MS?
It is a technique that combines high pressure liquid chromatography with tandem mass spectrometry to identify complex mixtures of proteins and peptides. It is used to identify proteins that are potential biomarkers, for drug development research, and in forensics to identify unknown compounds. Mass spectrometers can also be used for quality control and quality assessment, and to map structure of compounds.
What is 2D-DIGE?
Two Dimensional difference in-gel electrophoresis (2D-DIGE) is the most powerful variant of the classical two-dimensional gel electrophoresis, which adds simultaneous visualization and a highly accurate quantitative dimension to 2D gel electrophoresis. 2D-DIGE enables multiple protein extracts differentially labeled with spectrally resolvable CyDye fluors (typically Cy3 and Cy5) to be separated on the same 2D gel. By using a universal internal standard (equal amounts of all samples in the experiment) labeled with Cy2 multiple gels can be compared. Spots representing proteins-of-interest are identified, picked, digested, identified by LC/MS/MS and annotated.
What is The xMAP® technology?
The Multiplex Analyte Profiling (xMAPÂ®) technology was developed by the LuminexÂ® Corporation around proven flow cytometry, microspheres and traditional chemistry technologies. This technology features a flexible, open-architecture design and allows performance of a wide variety of bioassays accurately, quickly and cost-effectively. The technology allows simultaneous analysis (multiplexing) of up to 100 analytes in a single sample well through the combination of fluidics, optics, lasers, temperature control, software, high-speed digital processors and xMAP microspheres (beads). These 100 distinctly fluorescently dyed 5.6 micron-sized polystyrene beads serve as identifiers and a solid surface which enables performing a fast and cost-effective bioassay using relatively small sample volumes. Through the use of two lasers, the first red laser exciting the dye inside the bead and the second green laser exciting the fluorophore bound to the surface of the bead, the photo diode detectors can measure the excitation emission intensities inside the beads and a photomultiplier tube detects the excitation emission intensities on the surface of the bead. These intensity measurements are analyzed through high-speed digital processors and advanced computer algorithms, which allow for real-time analysis and quantification. This xMAPÂ® platform can be used for quantitative analysis of DNA, RNA and protein in biological and clinical samples.
How do I submit a request for analytical services?
Download and complete the Sample Submission Form available online at here.
– Submit completed form to ITSI-Biosciences via Email or Fax.
– Completed form will be reviewed and an initial Project Proposal presented to customer. This proposal will describe the exact services to be performed.
– The Project Proposal is then sent to the customer for approval.
– Customer reviews the proposal.
– Under normal circumstances, the project will not start until the proposed approach described in the Project Proposal is approved by the customer.
How should I ship my samples?
When the customer approves the Project Proposal, samples can then be submitted for analysis. Follow the instructions below for sample shipping.
– Make sure that the caps are tightly sealed on the sample tubes.
– Sample tubes can then be placed in either 15 mL or 50 mL conical tubes or in a zip lock bag for extra protection.
– Make sure that all the tubes are labeled appropriately.
– Half fill a Styrofoam box with dry ice.
– Add packaged samples in the box and top up with more dry ice to fill the container.
– Ship samples to ITSI-Biosciences for next day delivery, to the Attention of Dr. Richard Somiari (see full shipping address below).
– Send us the tracking number so we can track the package.
SUBMITTING SDS-PAGE GEL SLICES FOR ANALYSIS:
1. Care should be taken to minimize any possible keratin contamination by using clean instruments to cut out the gel slices and placing the slices in a clean microcentrifuge tube.
2. The gel slices should be soaked in ethanol, methanol, or acetonitrile for about 10 minutes to dehydrate the gel piece. After dehydrating the gel remove all the liquid, air dry and seal the tube.
3. To prevent the microcentrifuge tube from being damaged during shipping it should be packed in a larger tube or package; a 50ml conical centrifuge tube works well.
4. Once packed the gel slice can be shipped express delivery at room temperature.
SUBMITTING PROTEIN SOLUTION FOR ANALYSIS:
1. Precipitate proteins using either acetone or TCA precipitation method. Or you can use ToPrep kit.
2. Add 50 μl of ethanol to the precipitated proteins.
3. Follow step 3 from above.
FOR SHIPPING SAMPLES INTO THE UNITED STATES BY INTERNATIONAL EXPRESS:
1. Package the samples as above.
2. On the commercial invoice write the following in the description section:
SDS-PAGE gel slice for analytical testing. For Collaborative Research Purposes Only. Does not contain hazardous or infectious materials.
3. For the harmonized code (or tariff code) use: 3822.00.0002
4. For the value put $1.00
NOTE: Since shipping could be delayed due to unforeseen circumstances (such as weather), we advise that shipping should routinely be done on Monday or Tuesday to allow for samples to be received at ITSI on or before Friday.
633 Napoleon Street,
Johnstown, PA 15901
Attention: Dr. Richard I Somiari
- FAQ 2D-DIGE
- What type of samples can be analyzed?
- What type of buffer is suitable/best for 2D-DIGE experiments?
- What are the steps for the 2D-DIGE process at ITSI-Biosciences?
- What is the maximum number of samples that can be run on a 2D-DIGE gel?
- What is the amount of protein required to run a 2D-DIGE gel?
- What is the difference between 2D-Gel and 2D-DIGE?
- What is the turnaround time for 2D-DIGE analysis?
- Who owns the results generated?
- What happens to samples that are not completely used in the experiments?
- Does ITSI-Biosciences have a confidentiality policy regarding samples and results?
- What is the cost per sample for 2D-DIGE analysis?
- FAQ LC-MS/MS?
- How are the LC-MS/MS instruments used at ITSI-Biosciences?
- What kind of samples should be submitted to ITSI for protein identification?
- Does ITSI have the capability to quantify proteins by mass spectrometry?
- What is the smallest amount of protein that ITSI can detect?
- How am I confident that the identification is correct?
- What is the typical sequence coverage obtained?
- What typical turn around time?
- What kind of data do I get if I submit a protein sample?
- Do I get a discount if I submit multiple samples?
- How much is the cost of identifying a protein in a gel band?
- FAQ xMAP Technology?
- What is the xMAPÂ® technology?
- What are the xMAPÂ® microspheres (beads)?
- What are the benefits to xMAPÂ® technology?
- How does the Panomics QuantiGeneÂ® Plex 2.0 assay for RNA work?
- What kind of sample types are compatible with the Panomics QuantiGeneÂ® Plex 2.0 assay?
- How many RNA targets can be analyzed in each well for the Panomics QuantiGeneÂ® Plex 2.0 assay?
- Is it necessary to isolate or purify RNA prior to performing the Panomics QuantiGeneÂ® Plex 2.0 assay?
- How long does a gene expression profiling service take?
- How sensitive is the Panomics QuantiGeneÂ® Plex 2.0 assay?
- Are housekeeping genes required for the Panomics QuantiGeneÂ® Plex 2.0 assay?
- How many RNA samples can be run on the Panomics QuantiGeneÂ® Plex 2.0 assay?
- How many protein samples can be run on multiplex assay (xMAPÂ®)?
- What kind of assays currently exist?
- What types of samples are compatible with the multiplex assay (xMAPÂ®)?
- What is the cost of an xMAPÂ® or QuantiGeneÂ® assay?