2-D Electrophoresis
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Analytical Services
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Sample Preparation
It is important to remove salts, nucleic acids, polysaccharides and lipids when proteins are separated from the sample. Especially, salt must be removed using precipitation methods such as methanol precipitation, TCA/acetone precipitation or diafiltration by centricon. Otherwise, the sample-loaded strip is going to be burned.
For ideal protein analysis using MS compatible silver staining or Sypro-Ruby staining, 100 – 200 ug/gel of protein is required (for coomassie blue staining, 1 – 2 mg/gel) (normal gel: 18 cm). Sufficient amount of protein is required to do a pilot test before the actual experiment in order to confirm the optimal condition for separation.
| Roughly estimated amount (density) of protein samples | |||||||
| Bacteria | 50% of protein in the dry weight | ||||||
| Plasma/Serum | 60 mg/ml | ||||||
| 1×107 eukaryotic cell | 1 mg | ||||||
| Mammalian tissue | 1 mg of protein sample can be extracted from 10mg of lyophilized tissue | ||||||
| Urine | about 500 ml | ||||||
Samples for protein identification should be avoided keratin contamination.
Keratin is a hair and skin protein which occurs almost everywhere in dust and it can easily contaminate lab equipment and reaction tubes because of electrostatic charging. It will affect result critically.
Keratin is a hair and skin protein which occurs almost everywhere in dust and it can easily contaminate lab equipment and reaction tubes because of electrostatic charging. It will affect result critically.
1. At least 100ng and 3pmol of protein is needed for MALDI-TOF and LC coupled MS/MS analysis, respectively.
2. The diameter of gel spots or gel bands should be 1mm or less.
3. Using Ziptip is recommended for removing detergent, salt etc. and concentrating the sample.
4. Transfer your protein spot or band in an eppendorf tube without addition of any liquid.
5. Liquid samples should be shipped in a frozen state. Protein bands and protein spots can be shipped at room temperature.
2. The diameter of gel spots or gel bands should be 1mm or less.
3. Using Ziptip is recommended for removing detergent, salt etc. and concentrating the sample.
4. Transfer your protein spot or band in an eppendorf tube without addition of any liquid.
5. Liquid samples should be shipped in a frozen state. Protein bands and protein spots can be shipped at room temperature.
Gel staining
Coomassie Brilliant Blue Staining (CBB staining)
After 1-dimensinal electrophoresis or 2-dimensinal electrophoresis, the gels are stained. High sensitive staining solutions are required for obtaining reliable images. We are staining the gel with staining solutions those are prepared by our own methods. So we provide excellent images for analyzing the data.
CoomassiePRO (G250) is one of ProteomeTech’s product. This solution is high sensitive compared to conventional coomassie blue staining solution. We stain the gels with it and obtain excellent gel images. Furthermore,
the stained gels are easily destained with distilled water in a short time.
The protein spots or protein bands stained with CoomassiePRO are applicable to LC/MS and MALDI-TOF.
Silver Staining
We also stain the 2-DE or 1-DE gels with modified silver staining solution. The protein spots or protein gels stained with this silver staining solution are applicable not only LC/MS but also MALDI-TOF. The major feature of this solution is that there is little background in the gel.
Usually, the sensitivity of silver staining is much higher than that of coomassie blue staining as much as 10-100 times. Depend on the cases, very small protein spots or protein gels stained with silver staining are sometimes
very hard to be analyzed.
Image analysis
You can get several hundreds of protein spots with 2-dimensional electrophoresis. It takes long time to analyze the protein spots. But ProteomeTech use ImageMaster 5.0 (Amersham Biosciences) to analyze the protein spots and provide images and data. According to clients request, we provide several types of image analysis as following.
2-DE gel Premium analysis
All the proteins spots in the 2-DE gel are analyzed not only quantitatively but also qualitatively. ProteomeTech provides statistically processed data and you can use this data when submit journal and good for presentation.
2-DE gel Standard Analysis
We also provide only limited number of protein spot analysis according to clients’ request. You can save the cost when you want to know specific protein spots. The reliability of data is same to “2-DE gel premium analysis”.
1-DE gel quantitative analysis
We provide quantitative analysis of conventional SDS-PAGE protein bands. The expression of proteins are converted to relative numerical values and provided.
Isoelectric Focusing
It is known that all the proteins have their own isoelectric point (pI) values. Separation of proteins with their pI is first thing to do 2-dimensional electrophoresis.
It is called isoelectric focusing (IEF).
We use IPG (Immobilized pH Gradient) strip to separate pIs. The various pH ranged IPG strips are commercially available so you can separate the proteins in the
specific point in which you interest.
IPG DryStrip pH range (18㎝)
Broad range: pH3-10 (linear, non-linear), pH4 – 7, pH6 – 9
1 unit range: pH4.5 – 5.5, pH5.5 – 6.7
A lot of protein spots will be shown in pH3 – 10, but some proteins can be trailed in both end of IPG strip in many cases. We generally recommend pH4 – 7 range.
IEF Gel running
Depending on the customer’s request, ProteomeTech provides pI of specific protein. IEF gels (pH3 – 10, pH3 – 7), similar to conventional 1-DE gel, are used to investigate the pI value. IEF gels effectively create a pH gradient so proteins separate according to their unique pI. IEF Gels for pI determination are excellent for native applications using soluble proteins.
IEF Gel
SDS-PAGE
1DE
We offer conventional SDS-PAGE service. We perform SDS-PAGE to detect variable change of protein expression. Especially we perform 1-dimensional electrophoresis to confirm the sample concentration for 2-dimensional electrophoresis.
2DE
After separating the proteins using isoelectrofocusing, gradient polyacrylamide gel (usually 8-16%) is made to separate the proteins along its size.
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