Protein two-dimensional electrophoresis experimental service

1Protein two-dimensional electrophoresis experimental serviceIntroduction to Experimental Techniques

2D-WB is a technique that combines two-dimensional electrophoresis with traditional Western blot. The general experimental process is that the antigen protein mixture is separated by two-dimensional electrophoresis, then the gel protein is transferred to the support membrane, and then colored by antibody hybridization. 2D-WB technology can detect small changes in the isoelectric point or molecular weight of antigens, and has great applications in the study of protein post-translational modifications; The 2D-WB combined with mass spectrometry identification technology can identify antigens with stronger immunogenicity from protein mixtures.

IIProtein two-dimensional electrophoresis experimental serviceOverview

2D-WB is a technique that combines two-dimensional electrophoresis with traditional Western blot. The general experimental process is that the antigen protein mixture is separated by two-dimensional electrophoresis, then the gel protein is transferred to the support membrane, and then colored by antibody hybridization. 2D-WB technology can detect small changes in the isoelectric point or molecular weight of antigens, and has great applications in the study of protein post-translational modifications; The 2D-WB combined with mass spectrometry identification technology can identify antigens with stronger immunogenicity from protein mixtures.

3、 Service Content

1. Protein extraction and quantification;

2. Two dimensional electrophoresis;

3. Transfer membrane, seal, incubate, display imaging.

4、 Experimental operation process

1. The first item is electrophoresis (IEF), with gel strip equilibrium, and the second item is electrophoresis SDS-APGE

2. After the second electrophoresis, take out the gel for membrane transfer. The membrane transfer method can be wet transfer or semi dry transfer. The commonly used membranes are NC membrane and PVDF membrane.

3. Sealed, sealed with 5% skim milk powder or 5% BSA for 1 hour (room temperature) or overnight (4 degrees).

4. Primary antibody incubation: Select the incubation time according to the antibody instructions. The conventional incubation conditions are room temperature for 1 hour or overnight at 4 degrees Celsius. After incubation with the primary antibody, remove the membrane

Wash TBST three times, each time for 5 minutes.

5. Secondary antibody incubation: Select the appropriate secondary antibody (anti mouse, anti human, anti rabbit, etc.) based on the primary antibody, incubate at room temperature for 1 hour, then remove the membrane TBST and wash it 3 times, each time for 5 minutes.

6. Development: Dilute substrates A and B in proportion, mix them evenly on the mixing stage, and drop them onto the film. Place the film on the film in a dark room, cover it with a compression clamp, expose it for a certain period of time, and then put the film into the developing solution for development until clear protein bands appear. Rinse it with clean water and fix it in the fixing solution. The exposure time needs to consider factors such as the amount of protein loaded, antibody dilution concentration, and antibody incubation time.

7. After fixing, clean the film, air dry, calibrate the marker, scan the image, and analyze the 2D Western blot image.

Sample delivery instructions: In order to ensure the smooth operation of 2D Western blot technology services, sample delivery must meet the following requirements:

1. Customers provide: organization, cells, protein solutions, etc; First antibody (can be purchased by Yanjin Company).

2. Transportation requirements: Dry ice or liquid nitrogen packaging for shipping.

Note: Samples should avoid various types of contamination and repeated freeze-thaw cycles.

5、 Technical Service Report Content

1. Protein quantification results and electrophoresis sample size;

2. Original film, electronic images, and image analysis results;

3. Complete experimental report for 2D Western blot, including experimental steps, instrument and reagent usage, etc.

Attachment: 2D Western blot experimental steps

1. The main principles for preparing 2D Western blot protein samples are to dissolve all proteins as much as possible, disrupt protein-protein interactions, avoid various interfering substances, and ensure compatibility with IEF during sample preparation.

2. The main steps of two-dimensional electrophoresis include the first step of electrophoresis (IEF), gel equilibrium, and the second step of electrophoresis (SDS-APGE)

3. After the second electrophoresis, take out the gel for membrane transfer. The membrane transfer method can be wet transfer or semi dry transfer. The commonly used membranes are NC membrane and PVDF membrane.

4. Sealed, sealed with 5% skim milk powder or 5% BSA for 1 hour (room temperature) or overnight (4 degrees).

5. - Anti incubation: Select the incubation time according to the antibody instructions. The conventional incubation conditions are room temperature for 1 hour or overnight at 4 degrees Celsius. After incubation with the primary antibody, remove the membrane TBST and wash it 3 times, each time for 5 minutes.

6. Secondary antibody incubation: Select the appropriate secondary antibody (anti mouse, anti human, anti rabbit, etc.) based on the primary antibody, incubate at room temperature for 1 hour, then remove the membrane TBST and wash it 3 times, each time for 5 minutes.

7. Development: Dilute and mix substrates A and B in proportion, then drop them onto the film. Place the film on top of the film in a dark room, cover it with a compression clamp, and expose it for a certain period of time. Then, put the film into the developing solution for development until clear protein bands appear. Rinse with clean water and fix it in the fixing solution. The exposure time should consider the protein comprehensively Factors such as sample size, antibody dilution concentration, and antibody incubation time.

8. After fixing, clean the film, air dry, calibrate the marker, scan the image, and analyze the 2D Western blot image.

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