Frequently Asked Questions

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Check out our responses to some frequently asked questions below. If you have any more queries about our company, contact us.
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General FAQs

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.

 

Shipping Instructions

  • 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.

 

Shipping Address

ITSI-Biosciences
633 Napoleon Street,
Johnstown, PA 15901
Attention: Dr. Richard I Somiari

Does ITSI-Biosciences have a confidentiality policy regarding samples and results?

Yes. All experiments and transactions are confidential. ITSI-Biosciences will not use the results even for advertisement unless permission is given in writing by the owner. Every employee signs a legally binding non-disclosure and confidentiality agreement at the start of employment at ITSI-Biosciences.

LC/MS/MS FAQs

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 the structure of compounds.

How are the LC-MS/MS instruments used at ITSI-Biosciences?

Once a protein is identified ITSI will also help to figure out their role in biological systems. The LC-MS/MS instruments are complementary. The LC-MS/MS instrument is more sensitive, has a faster scan speed and can analyze many samples per day. The LC-MS/MS is primarily used for identification and quantitation of low abundant proteins, and sites of post-translational modifications can also be assigned to specific amino acid residues within a protein’s sequence using the LC-MS/MS.

What kind of samples should be submitted to ITSI for protein identification?

a) Identification and annotation,
b) Quantitation,
c) Post-translational modification mapping,
d) Verification of purity,
e) Detection of impurities in samples,
Both one-dimensional or two-dimensional gel bands stained with Coomassie, Silver, or Sypro stains can be submitted. The bands will be reduced and alkylated, digested with trypsin, and analyzed by LC-MS/MS. The MS/MS spectra generated during the analysis will be searched against theoretical spectra from protein databases such as the NCBI non-redundant database, the SwissProt database, and a number of databases from the International Protein Index using the SEQUEST algorithm. ITSI can also perform 1D gels, or comparative proteomics experiments using the 2D-DIGE technology, identify differentially expressed proteins, pick and digest candidate proteins with robots and identify the proteins.

Does ITSI have the capability to quantify proteins by mass spectrometry?

Yes. ITSI recently acquired a new mass spectrometer that is enabled for iTRAQ (Isobaric Tags for Relative and Absolute quantitation of proteins) the current benchmark for non-gel quantitative proteomics. With iTRAQ ITSI can simultaneously detect and quantify proteins. Either 4-plex or 8-plex experiments can be designed to allow for protein quantitation of up to 8 different sample types in a single analysis.

What is the smallest amount of protein that ITSI can detect?

At ITSI-Biosciences our systems are optimized to identify proteins at the attomole level. Any protein band that is visible by Coomassie stain will be identified in our laboratory.

How am I confident that the identification is correct?

A very stringent scoring system is used to identify peptides and proteins using the SEQUEST algorithm. Additionally, experienced mass spectrometrist’s with more than 20 years combined experience manually review the data before accepting the results.

What is the typical sequence coverage obtained?

The typical sequence coverage for “good and confident” protein identification is 30% of the protein’s entire sequence. Sequence coverage above 50% is considered great. At ITSI-Biosciences we routinely obtained sequence coverage as high as 85% for some proteins.

What is the typical turnaround time?

Turnaround time depends on what is required and the number of samples. Typically turnaround time for protein identification by LC/MS/MS is 48 hours for 1-10 samples. Turnaround time for Post Translational Modification mapping for 1-10 samples is about 5 days.

What kind of data do I get if I submit a protein sample?

You will receive a protein identification report that contains the a) sample ID, b) sample type, taxonomy, the names of the identified proteins, the accession numbers, the molecular weight and isoelectric point of the protein, the number of peptides matched to the protein, and the percent sequence coverage obtained for the protein. We also offer GO annotation including Molecular Function, Cellular Component and Biological Process of the identified protein.

Do I get a discount if I submit multiple samples?

Yes. A discount of 10 – 15% will be extended if more than 10 samples are submitted at a time. Please contact the mass spectrometry unit for more information/pricing.

How much is the cost of identifying a protein in a gel band?

The cost for protein identification depends on the sample complexity. Repeat customers and customers that submit multiple samples generally receive discounts.

2D-DIGE FAQs

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.

Multiple gels can be compared using a universal internal standard (equal amounts of all samples in the experiment) labeled with Cy2. Spots representing proteins of interest are identified, picked, digested, identified by LC/MS/MS, and annotated.

What type of samples can be analyzed?

Any sample from which proteins can be isolated can be analyzed. This includes all biological and clinical samples can be analyzed, such as blood, serum, plasma, tissue, cultured cells, culture media, tumor biopsies, and laser micro dissected samples. All animal, plant, nematode, microbial, insect and fish samples can also be analyzed.

What type of buffer is suitable/best for 2D-DIGE experiments?

Basically, any buffer system suitable for the upstream process can be accommodated. However, it may be necessary to include a precipitation and buffer exchange step. If samples are submitted in a different buffer then a buffer exchange step will be required prior to the 2D-DIGE process. This step may lead to unavoidable losses and changes in the protein content.

The 2D-DIGE process is very sensitive and the use of inappropriate buffer systems could affect the labeling efficiency, reproducibility and accuracy of the experiment. The best buffer for 2D-DIGE is the buffer system that is specifically prepared for 2D-DIGE. Based on more than 8 years experience we recommend the ToPI-DIGE (Total Protein Isolation Kit for 2D Difference Gel Electrophoresis) specifically formulated and validated for 2D-DIGE. This cost-effective buffer system available at https://www.itsibio.com/topi_dige.html reduces processing time and ensures reproducibility and quality results.

What are the steps for the 2D-DIGE process at ITSI-Biosciences?

Proteins are isolated from samples using ToPI-DIGE kit.
– If client sent samples already partially processed, then samples are precipitated to remove interfering substances (if necessary) and the samples are re-suspended in 2D-DIGE compatible buffer.
– Protein concentration is determined using the ToPA Bradford protein assay.
– If necessary, protein samples are qualified using the Agilent BioAnalyzer.
– 50µg of each protein is labeled with 200pmole Cy3 or Cy5 dye respectively, for the minimal labeling approach. If sample concentration is limiting, 30µg protein can be analyzed using the saturated labeling approach. Cy2 is used to label equal amounts of protein from all samples and used as the universal internal standard/control.
– The labeled samples are loaded on a 24cm IEF strip, pH 3-10. IEF strips with different (narrow and wide) pH ranges can be also used.
– The strips are rehydrated in the presence of the samples for 12 hrs at 30 volts.
– The rehydrated IEF strips are focused for a total of 65,000 volt hours.
– The focused strips are loaded onto a 24cm x 20cm, 12.5% SDS-PAGE gel, and run for 4 hours. The gel strength can be varied to select for low or high molecular weight proteins.
– The gels are scanned on a DIGE-enabled Typhoon Digital Imager at 3 different wavelengths.
– The images are analyzed using DeCyder analysis software (GE Healthcare). Depending on the experimental design, the statistical analysis is performed with the Difference In gel Analysis (DIA) module or the Biological Variation Analysis (BVA) module of DeCyder. The data obtained will be in p-values and ANOVA.
– Spots representing proteins-of-interest picked and tryptically digested with spot handling robots and identified by LC/MS/MS.

What is the maximum number of samples that can be run on a 2D-DIGE gel?

We can run up to three samples on one 2D-DIGE. Two samples will be labeled with Cy3 and Cy5 and the third sample labeled with Cy2 is the universal internal control that allows gel-to-gel comparison.

What is the amount of protein required to run a 2D-DIGE gel?

The minimum amount of an individual total protein sample recommended for a 2D-DIGE experiment is 30µg. However, we recommend sending at least 100µg of each sample. If protein amount is limiting then the “saturated” labeling approach is used. If protein concentration is not limiting then the “minimal” labeling approach is used. We have experience analyzing less than 30µg (as low as 5µg has been analyzed) but then only the high abundant proteins will be detected and downstream identification of a candidate spot by mass spectrometry becomes challenging due to the small amount of peptides that will be available for sequencing.

What is the difference between 2D-DIGE and Classical 2D(2D-PAGE)?

In the DIGE (Difference in-gel electrophoresis) process, protein samples are directly labeled with fluorescent dyes (typically Cy2, Cy3 and Cy5) before being separated by 2D electrophoresis. Since samples can be labeled with different fluorescent dyes they can be multiplexed on one gel, reducing gel to gel variations. In a 2D-PAGE only one sample per gel can be analyzed. Also to visualize a 2D- gel the gel must be stained, increasing the background and reducing sensitivity of detection, whereas in a 2D-DIGE gel the labeled proteins can be detected without staining the gel allowing low abundant proteins to be detected. With 2D-DIGE a few number of gels can be used for comparative proteomics.

What is the turnaround time for 2D-DIGE analysis?

The turnaround time for the 2D-DIGE aspect is 3 – 5 days for 10 samples or less. The turnaround time for an entire project that includes spot picking, in gel digestion and identification of the candidate proteins of interest by mass spectrometry is 2 – 3 weeks for most experiments.

Who owns the results generated?

All data generated are owned by the customer.

What happens to samples that are not completely used in the experiments?

Any sample not used in the experiment will a) be returned to the customer at the end of the experiment if requested (a fee will be charged for the shipping), b) immediately destroyed after the experiment, if directed by the customer or c) stored for up to 6 months with no charge to the customer if no instruction was received and space is available. At the end of 6 months the samples are discarded to make space or de-identified. Discarded or de-identified samples cannot be returned to customers.

What is the cost per sample for 2D-DIGE analysis?

ITSI-Biosciences price for 2D-DIGE process is very competitive and there are often special or seasonal promotions from the Dye manufacturer that can be passed on to customers. Since the cost will vary as a function of the experiment, we encourage clients to download and complete the Project Summary/Sample Submission Form available here to enable us better estimate the project.

Please contact us at: 1-814-262-7331 or itsi@itsibio.com if you have any more questions.

xMAP® Technology FAQs

What is xMAP® technology?

The Luminex® Corporation developed the Multiplex Analyte Profiling (xMAP®) technology around proven flow cytometry, microspheres, and traditional chemistry technologies. This technology features a flexible, open-architecture design and allows the performance of a wide variety of bioassays accurately, quickly, and cost-effectively.

The technology enables simultaneous analysis (multiplexing) of up to 100 analytes in a single sample 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, enabling 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 photodiode 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 xMAP® platform can quantitatively analyze DNA, RNA, and protein in biological and clinical samples.

What are the xMAP® microspheres (beads)?

The polystyrene beads are internally dyed with red and infrared fluorophores. In order to create 100 different microspheres, different quantities or ratios of the red and infrared dyes are implanted into the beads, giving each bead a unique spectral signature. Recently, magnetic beads have also been introduced. The magnetic beads called MagPlex Microspheres® by Luminex™ are super-paramagnetic microspheres that are internally labeled with fluorescent dyes and contain surface Carboxyl groups for covalent attachment of ligands (or biomolecules).

What are the benefits to xMAP® technology

Time and money savings through:
a) Multiplexing – Run up to 100 bioassays simultaneously in one sample well allowing for parallel analysis and efficient utilization of small amounts of samples.
b) Customized assays – Allow each scientist to develop assays for their individual needs.
c) Flexibility – Small sample amounts and multiple sample types allow assays to be tailored to the scientists’ requirements.
d) Cost/target – lower cost per target when compared to cost of performing PCR and ELISA for multiple targets.

How does the Panomics QuantiGene® Plex 2.0 assay for RNA work?

This assay combines branched DNA (bDNA) signal amplification technology, which amplifies the reporter signal instead of the target sequence, and xMAP® microspheres (beads) to provide accurate RNA profiling. First, RNA is released from the cells through cell lyses. Samples are then hybridized overnight, where mRNA transcripts are captured to the appropriate beads. After hybridization with a pre-amplifier and amplifier, Streptavidin-conjugated Phycoerythrin (SAPE) is bound to the mRNA transcripts bound to the bead. The lasers in the Luminex 200™ instrument measure the SAPE fluorescence, which is proportional to the amount of mRNA transcripts.

What kind of sample types are compatible with the Panomics QuantiGene® Plex 2.0 assay?

Cultured cells, whole blood, blood collected in PAXgene tubes, dried blood spots, fresh/frozen tissues, FFPE samples, and purified RNA can be analyzed.

How many RNA targets can be analyzed in each well for the Panomics QuantiGene® Plex 2.0 assay?

At this time up to 36 targets can be quantified in each well

Is it necessary to isolate or purify RNA prior to performing the Panomics QuantiGene® Plex 2.0 assay?

No, there is no need for RNA isolation/purification, reverse transcription, or amplification by polymerase chain reaction (PCR).

How long does a gene expression profiling service take?

Turnaround time depends on what is required and number of samples. Please call us for more details.

How sensitive is the Panomics QuantiGene® Plex 2.0 assay?

This assay has a Limit of Detection of ≤ 1,000-2,000 transcripts/assay well, a Limit of Quantitation of ≤ 2,000-4,000 transcripts/assay well and a Linear Dynamic Range of ≥ 3.5 logs.

Are housekeeping genes required for the Panomics QuantiGene® Plex 2.0 assay?

Housekeeping genes are not required. Although not required, it is recommended to include housekeeping genes, or control genes, since this allows for normalization.

How many RNA samples can be run on the Panomics QuantiGene® Plex 2.0 assay?

The Panomics QuantiGene® Plex 2.0 assay for RNA uses a 96 well plate, with 3 wells used for assay standards and background controls; therefore, 31 samples can be run in triplicates. More samples can be analyzed if single or duplicate assays are carried out.

How many protein samples can be run on multiplex assay (xMAP®)?

The multiplex protein assay uses a 96 well plate, with 9 wells used for assay standards and background controls; therefore, 29 samples can be run in triplicates. More samples can be analyzed if single or duplicate assays are carried out.

What kind of assays currently exist?

Multiplex assays are available for human, mouse, rat and monkeys. There are assays targeting multiple mechanisms and pathways. Off-the-shelf assays are available to quantify the following:

  • Cytokines
  • Angiogenic factors
  • Growth factors
  • Inflammatory and Acute phase proteins
  • Apoptotic proteins
  • Kinases
  • Neurobiology proteins
  • Cancer biomarkers

What types of samples are compatible with the multiplex assay (xMAP®)?

This assay can use the following sample types: tissue homogenate, whole blood, cell lysate, serum and plasma. Purified proteins and RNA can also be analyzed.

What is the cost of an xMAP® or QuantiGene® assay?

The cost for an assay depends on the type of target, number of targets and the number of samples that will be analyzed. If we know what you want to measure, we can provide you with a competitive quotation within 24 hours.

Testimonials

Hear what our happy customers have to say about us.

PhD Research Scientist, Department of Molecular Pharmacology, Division of Biochemistry, Walter Reed Army Institute of Research, Silver Spring, MD.

“I have used proteomic and biochemical analysis services and products offered by ITSI. I found those services received and products from ITSI outstanding and above my expectations. I will definitely recommend ITSI because of their quality, reliability and cost-effectiveness”.

PhD Research Scientist, Virginia Tech

“I have had good experience when I sought your help last time and now I’m planning to perform another experiment and would like to ask for a new quote”.

Throughput Operations Manager, Martin-Baker America, Johnstown, PA.

“Although, we’ve only used ITSI’s services briefly, I have found they have met our requirements to date. I have found the customer service to be very professional and detailed. Very pleasant to deal with. I do recommend ITSI. …as a DOD contractor, it’s important to build relationships with local businesses. A good deal for both of us”.

Instructor and Lead Faculty of Biological Sciences, Biology Club Advisor, Pennsylvania Highlands Community College, Johnstown, PA.

“ITSI exceeded my expectations. …our initial order went very well.… It makes sense to collaborate with others LOCALLY when you can and the prices were better than other companies”.