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Frequently Asked Questions

 

 

 

faq Get Started

faq Get Sample(s) Ready: Sample Type and Amount

faq Sample Shipping

faq About 2D DIGE Experiment

faq Phospho-Protein Profiling

faq About the Cost of 2D DIGE Experiment

faq Protein ID by Mass Spectrometry

faq Post 2D DIGE

QUESTIONS .........................................................................................................................................................................................................

Get Started:
1. How do I get start for my 2D DIGE experiments?

Get Sample(s) Ready: Sample Type and Amount
1. Can I submit cell pellets or tissues?
2. What lysis buffer should I use to extract protein?
3. How much protein is needed for each sample?
4. How many cells/tissue are needed for each sample?
5. Who is responsible for preparation of the protein samples?
6. How do I prepare immunoprecipitated (IP) samples for 2D DIGE?
7. Does Applied Biomics provide Immuno-Depletion of Serum Abundant Proteins?
8. Can I label only cell membrane proteins for 2D DIGE?

Sample Shipping
1. Where should I send the samples? What should I be aware of?
2. For sending international samples, what sample preparation method should I use?

About 2-D DIGE Experiment
1. What is the general process of 2D DIGE?
2. How many samples can I load on to one 2D DIGE gel?
3. What is the sensitivity of 2D DIGE? How many protein spots can you detect in the 2D DIGE experiment?
4. What is the difference between an Analytical Gel and a Prep-Gel?
5. What is the turnaround time for 2D DIGE?
6. What do I get from the data report?

Phospho-Protein Profiling
1. What is the Phospho-Protein Profiling?
2. Can the 2D DIGE experiment detect phosporylation?

About the Cost of 2-D DIGE Experiment
1. What is the cost for a typical 2DIGE/Protein ID experiment?

Protein ID by Mass Spectrometry
1. What is the general process of Protein ID by Mass Spectrometry?
2. What is cost for the spot picking?
3. What is the turnaround time for the Protein ID by Mass spectrometry?

Post 2-D DIGE
1. What follow up experiments can I do after 2D DIGE and Protein ID?
2. Can I do a Western blot after 2-D DIGE?
3. How can I do pathway analysis after the 2D DIGE/protein ID experiment?

ANSWERS ................................................................................................................................................................................................

Get Started

1. How do I get start for my 2D DIGE experiments?

You can send your samples (tissue, cell pellets, protein complex or protein extract) to Applied Biomics after filling out the order form if you are familiar with 2D DIGE. You are welcome to call or email Applied Biomics Technical Support (1-510-887-0889 or support@appliedbiomics.com) for assistance with 2D DIGE project design and sample prep instructions. Our tech support team will provide you with any requested information and assist you with customized 2D DIGE experiment design.

Get Sample(s) Ready: Sample Type and Amount of Samples

1. Can I submit cell pellets or tissues?

Yes, you can submit cell pellets and tissues. Cells must be washed several times with PBS to remove the medium. Tissues should be washed to remove potential blood contamination. Remove as much PBS as possible from the cell pellet or tissue prior to freezing in the labeled small plastic tube. All samples can be stored at -80 C before shipping in dry ice.

2. What lysis buffer should I use to extract protein?

We recommend using 2-D lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-HCl, pH 8.8) for protein extraction. If other buffers are used, you can replace the starting buffer with 2-D lysis buffer, or you can send us the protein extract, and we will exchange the buffer for 2-D lysis buffer here (free of charge). We prefer the protein concentration to be around 2 to 8 mg/ml. Samples in 2-D lysis buffer can be stored at -80 C for many months.

3. How much protein is needed for each sample?

Usually 25-50 ug of protein is needed for each sample in the analytical 2D DIGE gel. For the preparative 2D DIGE gel, about 150-300 ug of total protein is needed from each sample.

4. How many cells/tissue are needed for each sample?

Usually, about 2 million to 20 million cells are needed for the whole project. For tissues, we need about 2 mm x 2 mm x 1 mm pieces from each sample.

5. Who is responsible for preparation of the protein samples?

Applied Biomics will provide standard protein sample prep using 2D lysis buffer (7M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-HCl, pH8.8). You can send the tissues or cell pellets to us, and we'll extract the protein and measure protein concentration. For customized sample prep, please call our Tech Support (1-510-887-0889). For the immuno-depletion serum abundant protein service, additional charges will apply (Immuno-depletion of serum abundant protein service).

6. How do I prepare immunoprecipitated (IP) samples for 2D DIGE?

If you use IP in your experiments, please do the IP and the washing the same way you did before. At the end, please transfer the beads to a 1.5 ml plastic tube, and wash the beads once with 0.5 ml of water (this should get rid of the salt which may interfere with IEF). Please remove water as completely as possible after the final wash. The bead pellets can be kept at -20C and sent to us on dry ice. We will elute protein complex from the beads using a buffer that contains 2 M thiourea, 7 M urea, 4% CHAPS and 30 mM This-HCl pH 8.8.

7. Does Applied Biomics provide Immuno-Depletion of Serum Abundant Proteins?

Yes, Applied Biomics provides such service for serum samples. For human serum, we provide top 12 abundant protein depletion; for mouse and rat serum, we provide top 7 abundant protein immuno-depletion. See the Immuno-Depletion of Serum Abundant Protein for more details.

8. Can I label only cell membrane proteins for 2D DIGE?

Yes. You need to harvest fresh cells, and wash the cells with PBS (pH 8.0) before labeling with minimal CyDyes. To do this type of experiment, more minimal CyDyes is needed, and the cost will be higher. Please call our tech support to discuss the cost and the experiment design details.

Sample Shipping

1. Where should I send the samples? What should I be aware of?

The samples should be sent to:

Proteomics Service
Applied Biomics, Inc.
23785 Cabot Blvd. Suite 311
Hayward, CA 94545
Tel: 510-887-0889
Fax: 510-398-1515

Please fill out the order form with the sample information. For cells and tissues, please ship the package using Fedex or DHL with dry ice. Please also make sure to e-mail us the tracking number once you send out the samples, we'll track the package to make sure the samples are received. DO NOT send out the samples on Friday, as this could lead to dry ice evaporation and samples not received in good condition.

2. For sending international samples, what sample preparation method should I use?

For samples from countries outside the US, shipping time may be longer. Therefore, it's better to send protein extracts prepared through precipitation. Add 20 to 50 ul of 100% ethanol to the plastic tubes containing the protein precipitates. These samples can be sent by Fedex at room temperature, which would also save you the shipping cost by sending on dry ice.

About 2D DIGE Experiment

1. What is the general process of 2D DIGE?

Please click for an overview of the 2D DIGE work process. Protein samples are extracted from the cells or tissues, and protein concentration is measured and adjusted. The same amount of each protein sample is labeled with size and charge-matched minimal fluorescent CyDye. Up to three fluorescently labeled protein samples (for example, a normal control, a disease sample and a treatment sample) can be mixed and loaded onto the same electrophoresis gel. Most of our clients start with an analytical gel, on which small amount of labeled proteins are loaded (25-30 ug per sample, total protein is about 75-90 ug). This ensures that maximal resolution is achieved. The analytical images are excellent for publication and grant application purposes.

When changed protein spots of interest are identified from the analytical gel, a prep gel can be run to isolate the spots. About 600-700 ug of unlabeled proteins and a small amount of labeled proteins are loaded. This enables most protein spots we pick to be identified by Mass Spectrometry. Protein spots of interest are excised from the gel using a fully automated Ettan Spot Picker, and protein ID is determined from the mass spec analysis. The analytical gels take 1 week from the day we receive samples. Protein identification (including prep gel, spot picking and mass spec) takes another week.


2. How many samples can I load on to one 2D DIGE gel?

You can load up to three different samples on one 2-D DIGE gel. Thus you will have 3 paired in-gel sets of data for analysis: A versus B, B versus C and A versus C.

3. What is the sensitivity of 2D DIGE? How many protein spots can you detect in the 2D DIGE experiment?

The sensitivity of 2D DIGE is about 0.2 ng/spot. In general, using a pH 3-10 IPG strip, we will be able to detect over 2000 protein spots. If additional IPG strips were run using pH 4-7, pH 6-9 and pH 7-11, you will be able to detect over 5000 spots.

4. What is the difference between an Analytical Gel and a Prep-Gel?

Most customers request to start the experiment with an Analytical Gel for the following reasons:

1). Analytical gels use about 25 ug of each sample while prep-gels use about 250 ug of each sample. The total protein load for analytical gel is about 75 ug, while total protein load for prep-gels is about 750 ug. Since Analytical Gel contains lower sample amount resulting in less ion and salt in IEF, it will provide more accurate quantitation of protein changes) and much higher image quality for publication/presentation purposes.

2). After running the analytical gel, the in-gel data analysis will be done. Based on the analytical gel results, you can put more control or treatment samples if your desired spots are from control (usually down-regulation), or from treatment (usually up-regulation or newly induced proteins). In contrast, Prep-gel is much less likely to provide such information due to the excess amount of protein.

3). From the analytical gel, you will know if you need to focus on a specific region (i.e. with most changes) in the next 2-D experiment by changing the pH range for IEF or the percentage of the SDS-gel (for resolving lower or higher MW protein spots). In contrast, Prep-gel is much less likely to provide such information due to the excess amount of protein.

4). The main purpose of running Prep-gel is to obtain enough protein amount for the protein ID by Mass Spectrometry.

5. What is the turnaround time for 2D DIGE?

The turnaround time for 2-D DIGE is 7 days after we receive the samples; the turnaround time for Protein ID by Mass Spectrometry is 7 -10 days.

6. What do I get from the data report?

Once the data report is ready, we will e-mail you the link for downloading your data report. The data report contains two main parts:

1) ImageQuant analysis: gel images of individual samples and overlay of two sample images;

2) DeCyder DIA Analysis: quantitative analysis and comparison of all spots between different samples. The differential protein expression is assessed by Differential In-gel Analysis (DIA), giving the high accuracy and reproducibility.
For your convenience, we will also make recommendations on follow-up spots with quantitative data and locations. See the Sample Report.

Phospho-Protein Expression Profiling

1. What is the Phospho-Protein Expression Profiling?

Phospho-Protein Profiling is to detect phosphorylated protein spots in the 2D gel. One bonus aspect is that there is no need for phospho-antibodies, and no need for radioisotope labeling. We provide 2 services in this area:

a) 2D DIGE Followed by Phospho-protein Profiling: you will be able to find the phospho-protein spots and their quantitative changes between control and treated samples (see an example).

b) Total Protein versus Phosphorylated Protein on 2D Image: both will be viewed on the same gel.

2. Can the 2D DIGE experiment detect phosporylation?

Yes. When proteins are phosphorylated, the protein pI will shift to a more acidic region and it can be easily detected by 2D DIGE. For identification of phosphorylation site, you will need more protein and different methods such as phospho-peptide enrichment, LC-MS/MS etc.

About the Cost of 2D DIGE Experiment

1. What is the cost for a typical 2DIGE/Protein ID experiment?

Let's assume a new customer from academics who is interested in finding the differential protein expression among 2-3 samples, followed by obtaining Protein ID for the top 10 spots. Please note that the prices are based on gels, not on each sample. Each gel can load up to 3 samples. Following would be the estimated cost:

2 samples
3 samples
Analytical Gel
$1050*
$1195*
Preparatory Gel
$1200
$1295
Spot Pick (up to 96 spots)
$250
$250
10 Protein ID by Mass Spectrometry
$1150
$1150
Total cost
$3650
$3890

 

* Only applies for new customers who submit standard protein samples with protein concentration >5mg/ml. The discount is subject to approval from Finance. The discounted price applies for the first 2D DIGE Analytical gel. The discounted price does NOT apply for Preparative gels.

Protein ID by Mass Spectrometry

1. What is the general process of Protein ID by Mass Spectrometry?

It includes the following steps:

1). Gel treatment: Gel is washed multiple times to remove staining dye and other chemicals inhibitory to Mass Spectrometry

2). In-gel trypsin digestion: Trypsin digestion buffer is added and protein is digested in gel at 37°C

3). Peptide extraction: Digested peptide sample is extracted from gel by extraction buffer

4). Desalting: The digested tryptic peptides are desalted by C-18 Zip-tip (Millipore)

5). Spotting: Sample is mixed with matrix buffer and spotted on MALDI plate

6). MALDI-TOF: MS spectra are obtained using Applied Biosystems proteomic analyzer

7). MALDI-TOF/TOF: Ten to twenty most abundant peptides are further subjected to fragmentation (MS/MS)

8). Database search: Both MS and MS/MS spectra are submitted for database search using GPS explorer equipped with MASCOT search engine

2. What is cost for the spot picking?

There is a flat set-up fee for spot picking. The cost is $200/gel for up to 96 spots.

3. What is the turnaround time for the Protein ID by Mass Spectrometry?

The turnaround time for the Protein Identification by Mass Spectrometry service is 7 days for spots picked from our 2D-DIGE service. For protein/gel spots submitted from external sources, the turnaround time is 7-10 days. Please provide information on the species of your sample so that we can search the correct database.

Post 2D DIGE

1. What follow up experiments can I do after 2D DIGE and Protein ID?

In general, after 2-D DIGE and Protein identification of differentially expressed spots, the following methods can be used to obtain additional detailed functional information about the proteins.

1). Pathway analysis (by Ingenuity Pathway Analysis, or GeneGO Pathway Analysis) to determine which kinase interacts with or regulates the identified proteins, and which drugs regulate this pathway. In general, the more proteins identified, the better the chance of identifying the major pathway.

2). Western blot or Northern blot to confirm the identified expression changes (including the ones found by pathway analysis).

3). Immuno-staining to confirm the identified expression changes (including the ones found by pathway analysis).

4). Change the experiment conditions, and confirm the targeted functional protein changes.

2. Can I do a Western blot after 2D DIGE?

Yes. After 2D DIGE gel scanning, we can transfer the proteins from the gel to PVDF membrane, and send the membrane to you for Western Blotting. We also provide the Western blotting service if you send us the primary antibodies.

3. How can I do pathway analysis after the 2D DIGE/protein ID experiment?

Once you have finished the 2D DIGE/protein ID experiments, we can perform a cluster analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Please call our tech support and we can provide additional information.