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(1) Source of primary antibody species
The type of secondary antibody is mainly determined according to the source of the primary antibody species. If the primary antibody is derived from a mouse, then the secondary antibody can be purchased from anti-mouse (such as sheep anti-mouse, rabbit anti-mouse, etc.).
(2) Type of primary antibody
The secondary antibody must match the category or subclass of the primary antibody. This is usually for monoclonal antibodies. Polyclonal antibodies are mainly IgG immunoglobulins, so the corresponding secondary antibodies are anti-IgG antibodies.
The types and subclasses of monoclonal antibodies are usually described in the product instructions. If your primary antibody is mouse IgM, then the corresponding secondary antibody should be anti-mouse IgM.
If the monoclonal primary antibody is a subclass of mouse IgG (IgG1, IgG2a, IgG2b, IgG3), then almost all anti-mouse IgG can be combined with it, or you can choose a specific one for this subclass Secondary antibodies, for example, if your primary antibody is mouse IgG1, then you can choose a secondary antibody against IgG1, which is especially suitable for double labeling experiments.
When it is not clear what kind / subclass of primary antibody, anti-IgG of the corresponding species can be used.
(3) Selection of markers
Generally speaking, the probes coupled to the secondary antibodies mainly include enzymes (horseradish peroxidase HRP and alkaline phosphatase AP or its derivatives APAAP, PAP), fluorophores (FITC, RRX, TR, PE) And biotin. The choice of probe secondary antibody depends on the specific experiment. For western blot and ELISA, the most commonly used secondary antibodies are enzyme-labeled secondary antibodies, and fluorescent or labeled secondary antibodies are usually used in cell or tissue labeling experiments (cytoimmunochemistry, tissue immunochemistry, flow cytometry). A secondary antibody labeled with horseradish peroxidase or alkaline phosphatase can also be used in the chemical conversion. If you want to amplify the detection signal to a greater degree, you can use the biotin / avidin detection system.
The following briefly introduces the advantages and disadvantages of several secondary antibody probes and their applicable experiments:
a. Enzyme:
There are two main types of enzyme conjugates, HRP and AP. In comparison, the former is more economical, fast, and stable, while the latter is more sensitive than the former. Generally, HRP is widely used in immunohistochemistry, western blot and ELISA, and alkaline phosphatase (AP) is more suitable for solid Phase immunoassay experiments, such as ELISA and western blot.
b. Fluorescent group:
FITC: FITC is a widely used fluorophore, but its biggest disadvantage is its fast quenching, which can be alleviated by the use of anti-quenching agents.
AMCA: Commonly used in multi-label experiments, such as immunofluorescence and flow cytometry. The disadvantage is that the quenching is fast, and because the blue fluorescence excited by the naked eye cannot be well detected, the AMCA-coupled secondary antibody is more suitable for detecting a large number of antigens.
Cyanine dyes (Cy2, Cy3, Cy5): Cy2 is more stable than FITC, and under surface fluorescence microscope, Cy2 has stronger FITC fluorescence. Cy3 and Cy5 are brighter, more stable, and have a weaker background than most fluorophores. Both are often marked in confocal microscopy experiments. However, the disadvantage of Cy5 is that it cannot be observed with a surface fluorescence microscope, so it is usually observed with a confocal microscope with a far infrared detector.
TRITC, RRX, TR: TRITC is often used with FITC in dual-label experiments. RRX or TR can also be used, but TR will produce a higher background. RRX is especially useful when using laser confocal scanning microscope equipped with a krypton hydrogen lamp for three-mark experiments. It can be used with Cy2 (or FITC) and Cy5 because the emission wavelength of RRX is between Cy2 and Cy5, and the two There is very little overlap.
PE: often used with FITC for double-labeled flow cytometry experiments.
Note: Since the observation of fluorescence requires a certain wavelength of excitation light, it must be selected according to the wavelength band that the existing instruments in the laboratory can emit light.
Fluorescein can be divided into:
<499nm, blue fluorescence (Blue)
500-549nm, green fluorescence (Green)
550-584nm, yellow fluorescence (Yellow)
585-615nm, orange fluorescence (Orange)
616-700nm, red fluorescence (Red)
> = 700nm, far-infrared fluorescence (Far-Red)
Several new fluorescent groups
Fluorophore
colour
Excitation light wavelength (nm)
Wavelength of emitted light (nm)
DyLight 488
493
518
DyLight 549
550
568
DyLight 649
646
674
DyLight 680
Near Red
682
715
DyLight 800
Infrared
770
794
n Higher fluorescence brightness
n Luminescence is more durable, suitable for fluorescent photography
n Higher sensitivity
n High SNR and low background
n Stability is better for various buffer systems with pH 4-9
c. Biotin:
Biotin can bind closely with antibiotic protein / streptavidin (avidin / streptavidin). First add the biotin-labeled secondary antibody, and add avidin or streptavidin coupled with an enzyme or fluorescent group, the latter can bind to multiple sites on the surface of the secondary antibody, thereby greatly amplifying the detection signal.
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