FAQs
2-D Gel Electrophoresis
How long should I expect to wait for the lab results after the beginning of the analysis?
What quantity of proteins is needed for the analysis?
How should the samples be prepared?
How do I interpret my analysis report?
Protein Identification
How long should I expect to wait for the lab results after the beginning of the analysis?
What quantity of proteins is needed for the analysis?
How should the samples be prepared?
How do I interpret my analysis report?
Why are the proteins in my sample not identified?
What is the difference between MALDI-TOF-TOF MS on ABI 4800 and LC/MS/MS on LTQ Orbitrap Velos for protein identification?
What is the difference between mass spectrometry and Western blotting for protein identification?
Why do I see keratin in my samples?
Why do I see albumin and casein in my gel-purified samples?
In the analysis report, I see a list of proteins from Mascot or GPS search. Are all these proteins in my samples?
Protein Quantification
1. ICAT
How long should I expect to wait for the lab results after the beginning of the analysis?
What quantity of proteins is needed for the analysis?
How should the samples be prepared?
How do I interpret my analysis report?
What are the differences between using ICAT and iTRAQ for protein identification?
2. ITRAQ
How long should I expect to wait for the lab results after the beginning of the analysis?
What quantity of proteins is needed for the analysis?
How should the samples be prepared?
How do I interpret my analysis report?
What are the differences between using ICAT and iTRAQ for protein identification?
Protein post-translation modifications
How long should I expect to wait for the lab results after the beginning of the analysis?
What quantity of proteins is needed for the analysis?
How should the samples be prepared?
How do I interpret my analysis report?
Why do you need a larger quantity of proteins for phosphorylation site mapping than for identification?
What steps are necessary for mapping protein phosphorylation sites?
Collaboration, experimental design and other questions
How is collaboration different from fee-based services?
What costs are associated with collaboration?
Whom should I contact to ask questions regarding experimental design?
Whom should I contact to ask questions regarding data interpretation?
Whom should I contact to ask questions regarding billing?
Whom should I contact to ask questions regarding sample shipment and reception?
2-D Gel Electrophoresis
How long should I expect to wait for the lab results after the beginning of the analysis?
2 weeks
Top of Page
What quantity of proteins is needed for the analysis?
100 mg for samples that DO NOT need further cleanup or buffer exchange.
250 mg for samples that DO need further cleanup
Top of Page
How should the samples be prepared?
You can submit cell pellets or tissues that are cleaned from blood. Keep the samples low in salt and detergent buffers.
Top of Page
How do I interpret my analysis report?
Using the analysis report, look at the provided gel images. Horizontally, the graph is distinguished by isoelectric point (PI).Vertically, the graph is distinguished by size (MW).Compare results with the MW markers provided on the left side of the graph.
Top of Page
Protein Identification
How long should I expect to wait for the lab results after the beginning of the analysis?
2 weeks
Top of Page
What quantity of proteins is needed for the analysis?
No less than 50 ng/ band
Top of Page
How should the samples be prepared?
Gel samples—Submit the samples either as intact gels or excised bands or 2D gel spots in <5 mL of water. Regarding staining, Coomassie blue, SYPRO Ruby, and other fluorescent dyes are acceptable. However, DO NOT use silver stain to stain your protein samples.
Solution samples—Send in a properly sanitized container. Please provide us the buffer compositions and protein concentration assay results prior to sample submission.
Top of Page
How do I interpret my analysis report?
Upon receiving your analysis report (see example results page), direct your attention to the accession numbers printed next to the protein name. Use that number to retrieve the protein sequence from an online protein data bank (http://www.ncbi.nlm.nih.gov scroll down to “protein” under search menu), you can identify the specific protein and get other information about the protein. On occasion, the protein name may not be informative. In this case, you can find similar proteins via Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
Top of Page
Why are the proteins in my sample not identified?
There are numerous reasons why your proteins have not been identified. The most common are due to:
1. The sample amount provided was insufficient;
2. The protein sample had not been purified adequately;
3. There was too great a mixture of proteins in the sample in which the target protein was too low to be identified;
4. The solution sample has been contaminated by keratin.
Top of Page
What is the difference between MALDI-TOF-TOF MS on ABI 4800 and LC/MS/MS on LTQ Orbitrap Velos for protein identification?
MALDI-TOF-TOF is an instrument used to identify proteins in simple, pure samples (such as gel bands) with low complexity.
LC/MS/MS is a machine used to identify proteins in complex solution samples.
Top of Page
What is the difference between mass spectrometry and Western blotting for protein identification?
MS can identify unknown proteins. With the results, one is able to identify proteins from the protein sequence databases. Western blotting uses a known antibody to confirm that the protein is in fact the one that is located in the sample gels. WB can be performed with 1D or 2D gels, and it is typically more sensitive than MS for protein identification.
Top of Page
Why do I see keratin in my samples?
Keratins are everywhere; human skin flakes and human hair are the most probable reason for predominant keratin results. Make sure that you keep samples uncontaminated by using powder-free gloves, keeping samples shielded from outside contaminants, refraining from touching the sample with bare hands, and avoiding community reagents. Also, clean lab glassware or plastic wares with bleach prior to using them. Try to use dedicated lab wares for protein chemistry. Do not use recycled reagents, dyes, etc.
Top of Page
Why do I see albumin and casein in my gel-purified samples?
If the sample came from a cell culture or your sample was a tissue sample that still had blood on it, your sample will have albumin contamination. If the sample was housed in a container that was previously used for western blotting, it may contain casein, an abundant milk protein.
Top of Page
In the analysis report, I see a list of proteins from Mascot or GPS search. Are all these proteins in my samples?
Not all of them. Primarily, look for protein hits that have 2 or more peptides matched with confidence interval (C.I.) values that are greater than 95%. You need to use rational judgment to determine which protein is correct. Take into consideration the sample source.
Top of Page
Protein Quantification
1. ICAT
How long should I expect to wait for the lab results after the beginning of the analysis?
1 month
Top of Page
What quantity of proteins is needed for the analysis?
200 micrograms
Top of Page
How should the samples be prepared?
Solution should not contain DTT, TCEP, mercaptoethanol, or other reducing agents.
Top of Page
How do I interpret my analysis report?
You will be provided with a list of cysteine containing peptides with ICAT H/L ratio with changes of at least 20% from the population mean.
Top of Page
What are the differences between using ICAT and iTRAQ for protein identification?
In ICAT, the peptides are labeled on cysteine residues and they are the only ones quantified. It is also useful for determining changes in protein redox states. In iTRAQ, peptides are labeled at the N-terminal and free amines of lysine residues. In general, more proteins are quantified by the iTRAQ method.
Top of Page
2. ITRAQ
How long should I expect to wait for the lab results after the beginning of the analysis?
6 weeks
Top of Page
What quantity of proteins is needed for the analysis?
50-100 mg in 50 mL of iTRAQ buffer
Top of Page
How should the samples be prepared?
The buffer should not contain ionic detergent or primary amines (NH3). Refer to http://tools.invitrogen.com/content/sfs/manuals/ITRAQprotocol_man.pdf.
Or search “iTRAQ’s Reagent Protocol” on invitrogen website.
Top of Page
How do I interpret my analysis report?
You will be provided with a list of proteins which are significantly (p-value 0.05) changed (with a fold cut off of ±20% from the population mean). Each protein should contain 2 or more peptides with C.I. value of 95% or better.
Top of Page
What are the differences between using ICAT and iTRAQ for protein identification?
In ICAT, the peptides are labeled on cysteine residues and they are the only ones quantified. It is also useful for determining changes in protein redox states. In iTRAQ, peptides are labeled at the N-terminal and free amines of lysine residues. In general, more proteins are quantified by the iTRAQ method.
Top of Page
Protein post-translation modifications
What kind of protein post-translation modifications have you been able to detect?
Phosphorylation, methylation, acetylation, citrullination, hypusination, nitrosylation
Top of Page
How long should I expect to wait for the lab results after the beginning of the analysis?
1 month
Top of Page
What quantity of proteins is needed for the analysis?
10 mg of pure protein
Top of Page
How should the samples be prepared?
Gel samples—Keep sample within intact gels or excised bands or spots in <5 mL of water. Regarding staining, Coomassie blue, SYPRO Ruby, and other fluorescent dyes are acceptable. However, DO NOT use silver stain to stain your protein samples.
Solution samples—Send in a properly sanitized container. Please provide us the buffer compositions and protein concentrations prior to sample submission.
Top of Page
How do I interpret my analysis report?
Look for the MS/MS sequence identification ions in the spectrum and MS/MS ions including fragment ions encompassing the phosphorylation site.
Top of Page
Why do you need a larger quantity of proteins for phosphorylation site mapping than for identification?
For phosphorylation site mapping, less than 5% of proteins are phosphorylated, so certain phosphor-enrichment steps are required before phosphorylation site mapping can occur.
Top of Page
What steps are necessary for mapping protein phosphorylation sites?
Protein digestion, phosphor-peptide enrichment, LC-MS/MS analysis, database search, manual De Novo sequence for phosphorylation site identification. On occasion, alternative protease digestions other than trypsin are needed to obtain sufficient sequence coverage to identify phosphorylation sites.
Top of Page
Collaboration, experimental design and other questions
How is collaboration different from fee-based services?
Fee-based services are straight forward processes. The customer provides the samples, we provide the results, and the payment is submitted. If there is any extra consultation needed, an extra fee will be charged.
Collaboration is the preferred method when the project is more complex and requires more back and forth dialogue regarding data analysis and experimental design. This service also includes aid in grant preparation, manuscript submission, and figure preparation. Authorship will be requested, as well as percent effort allocation in grant applications.
Top of Page
What costs are associated with collaboration?
The cost is predetermined before collaboration type projects begin. No itemized fees will be charged.
Top of Page
Whom should I contact to ask questions regarding experimental design?
Dr. Hong Li (primary) - 973-972-8396 or liho2@umdnj.edu
Dr. Tong Liu (secondary) - 973-972-5340 or linto@umdnj.edu
Top of Page
Whom should I contact to ask questions regarding data interpretation?
Dr. Hong Li - 973-972-8396 or liho2@umdnj.edu
Dr. Tong Liu - 973-972-5340 or linto@umdnj.edu
Top of Page
Whom should I contact to ask questions regarding billing?
Dr. Hong Li - 973-972-8396,
Dr. Qing Li - 973-972-5340 or liq3@umdnj.edu
Top of Page
Whom should I contact to ask questions regarding sample shipment and reception?
Dr. Qing Li - 973-972-5340 or liq3@umdnj.edu
Dr. Tong Liu - 973-972-5340 or linto@umdnj.edu
Top of Page
| 
