SILAC Metabolic Labeling Quantitative Experimental Service

SILAC Metabolic Labeling Quantitative Experimental Service

Introduction to Experimental Techniques: The basic principle of SIL AC is to replace the corresponding amino acids in the cell culture medium with natural isotopes (light) or stable isotopes (heavy) labeled essential amino acids. After 5-6 doubling cycles, the stable isotope labeled amino acids * are incorporated into the newly synthesized protein of the cell to replace the original amino acids. The lytic proteins of different labeled cells are mixed in proportion to the number of cells or protein content, separated and purified, and identified by mass spectrometry. Relative quantification is performed by comparing the area of two isotopic peptide segments in the primary mass spectrum. It belongs to the in vivo metabolic labeling method.

Experimental operation process:

1. Protein extraction and sample preparation

2. Marking efficiency test

3. Sample mixing table

4. SDS-PAGE grading

5. Enzymatic hydrolysis

6. Mass spectrometry identification

7. Software analysis

8. Qualitative and quantitative proteomic results

9. Bioinformatics Data Mining and Statistical Analysis

Experimental precautions:

The customer provides:

1. Protein extract: Concentration>1 μ g/ul, total protein content>300 μ g

2. Cells: Take at least 2x106 cells by cell counting and transfer them to an EP tube. Add 0.5ml of physiological saline or protein protectant, mix well, and store in a refrigerator (-80 ° C to avoid repeated freezing and thawing).

Delivery standards:

1. Protein identification and SIL AC quantitative analysis results;

2. Customer pointingThe mass spectrometry peak corresponding to the protein;

3. The complete experimental steps, instrument usage, software retrieval parameters, etc. of SIL AC experiment.

Others

1. SILAC is an in vivo labeling technique, in which stable isotope labeled amino acids have similar chemical properties to natural amino acids. The labeled cells and unlabeled cells exhibit almost no difference in biological behavior, and the labeling efficiency can reach up to 100%.

2. Using mass spectrometry for quantification, the quantitative results are accurate with minimal batch variation and good repeatability.

3. The combination of in vivo metabolic markers with SDS-PAGE or chromatographic separation techniques is compatible with hydrophobic and alkaline proteins, and is not limited by protein properties.

4. After mixing multiple samples, separation, enzyme digestion, and identification were carried out simultaneously. Subsequent experiments had consistent effects on the samples, reducing the impact of experimental operations and equipment.

5. As this technology belongs to in vivo labeling, the labeling effect is stable, and its labeling efficiency is not affected by lysis buffer. It is not only suitable for the analysis of whole cell proteins, but also for the identification and quantification of membrane proteins.

6. Compared with chemical labeling, the SIL AC method requires significantly less protein, typically only a few tens of micrograms of protein per sample.

7. Live labeling, closer to the true state of the sample.

8. It is only applicable to cells cultured in vivo and cannot be analyzed for commonly used tissue samples, body fluid samples, etc. in biomedical research. The labeling cost for animal models is too high to achieve. Compared with in vitro labeling techniques, SIL AC belongs to in vivo labeling and has the following technical advantages

1. High throughput, capable of simultaneously identifying and quantifying hundreds to thousands of proteins;

2. Quantitative precision *&accuracyReduce experimental differences caused by different sample preparation methods;

3. Wide linear quantitative range;

4. Higher sensitivity and significantly reduced protein requirement;

5. The labeling adopts in vivo labeling technology, which is closer to the true state of the sample.

SILAC technical service content

1. Cell isotope labeling;

3. SDS-PAGE separation;

4. Liquid phase separation and mass spectrometry analysis after trypsin digestion;

5. Database retrieval and protein quantitative analysis;

6. Bioinformatics analysis of differential proteins;

7. Experimental report submission.

Content of Experimental Service Report

1. Protein identification and SIL AC quantitative analysis results;

2. Customer pointingThe mass spectrometry peak corresponding to the protein;

3. The complete experimental steps, instrument usage, software retrieval parameters, etc. of SIL AC experiment.

Technical service advantages

1. High sensitivity, low detection limit, capable of detecting low abundance proteins;

2. IITRAQ has strong separation ability and a wide analysis range. It can separate and identify any type of protein, including high molecular weight proteins, acidic and alkaline proteins, membrane proteins, and insoluble proteins;

3. High throughput: Simultaneously analyzing 8 samples improves experimental throughput, allowing for simultaneous analysis of proteins at multiple time points or different treatments;

4. Reliable results: Qualitative and quantitative analysis results are more reliable;

5. High degree of automation: liquid phase and mass spectrometry combined, automated operation, fast analysis speed, and good separation effect.

Experimental outsourcing service: