Get
Started
Get
Sample(s) Ready: Sample Type and Amount
Sample
Shipping
About
2D DIGE Experiment
Phospho-Protein
Profiling
About
the Cost of 2D DIGE Experiment
Protein
ID by Mass Spectrometry
Post
2D DIGE
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
dedect 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?
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-deplietion
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: 510-887-0867
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 Spectromety.
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
Spectromety 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 dedect 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, following would be the estimated cost:
|
|
2 samples
|
3 samples
|
|
Analytical Gel
|
$795 (trial discount)
|
$940 (trial discount)
|
|
Preparatory Gel
|
$795 ( trial discount)
|
$940 (trial discount)
|
|
Spot Pick (up to 96 spots)
|
$200
|
$200
|
|
10 Protein ID by Mass Spectrometry
|
$1150
|
$1150
|
|
Total cost
|
$2,940
|
$3,230
|
Please note that the prices are based on gels, not on
each sample. Each gel can load up to 3 samples.
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,
you can search for pathways by using different pathway analysis
software to get more information. We usually use Ingenuity
or GeneGo software. Please call our tech support and we
can provide some help.
