Immunofluorescence localization experiments of nuclear proteins
Immunofluorescence localization of nuclear proteins can be applied to (1) localize intracellular distribution of nuclear proteins and (2) viral studies.
Operation method
immunofluorescence localization method
Principle
Immunofluorescence cytochemistry is the use of fluorescein-labeled known antibody (or antigen) as a probe to detect the target antigen (or antibody) in the tissue and cell specimen to be tested, the formation of antigen-antibody complex with fluorescein, under a fluorescence microscope, due to the high-pressure mercury lamp source of ultraviolet irradiation, the fluorescein emits a bright fluorescence, so that you can distinguish the location of the antigen (or antibody) and its properties Under the microscope, fluorescein emits bright fluorescence due to ultraviolet light irradiation from a high-pressure mercury lamp, so that the location and nature of the antigen (or antibody) can be distinguished and the content of the antigen can be calculated by fluorescence quantification technology, in order to achieve the purpose of localization, qualitative and quantitative determination of antigenic substances.
Materials and Instruments
Nucleoprotein Move 1. Cultivate cells on 22x22 mm No. 1.5 coverslips. Ideally cells should grow to 50% to 70% confluence. Caveat 1. mark the corners of one side of the coverslip to know that the cells are on that side of the coverslip. 2. The concentration of primary antibody should be determined by experiment. 3. if a 22 x 22 mm coverslip is used in step 4, add 30 ul of diluted antibody to the coverslip and invert the coverslip onto a glass slide, then place the slide in a wet box and incubate at room temperature. Common Problems Nuclear proteins are proteins that are synthesized in the cytoplasm and then transported to the nucleus. Such as a variety of histones, DNA synthetases, RNA transcription and processing enzymes, a variety of regulatory protein factors. Nuclear proteins generally contain special amino acid signal sequences, which play a role in protein orientation and localization. For more product details, please visit Aladdin Scientific website.
Methanol PB Primary antibody Secondary antibody
Microscope Slide Coverslip Dropper
2. Fix cells with 100% methanol at -20°C for 3 minutes.
3. Wash three times with PBS + 1% NGS for 10 minutes each time.
4. incubate in a wet box for 1 hour at room temperature with the appropriate concentration of primary antibody.
5. Wash three times with PBS + 1% NGS for 10 minutes each time.
6. incubate in a wet box with fluorescently labeled secondary antibody diluted at 4 ug/ml for 1 hour at room temperature.
7. Wash four times with PBS for 10 minutes each.
8. Seal the coverslip on the carrier sheet with sealer and seal the edges of the coverslip with clean nail polish to prevent the coverslip from sliding.
Source: Cell Biology Laboratory Manual