The following article presents quick tips on how to choose secondary and protocol optimization for fluorescent western blot.
Choosing Your Secondary
To achieve clear and bright bands, an optimized antibody for fluorescent western blot is needed.
- When multiplexing, it is recommended when choosing individual primary antibodies that each of them is from a different species.
- To minimize cross-species reactivity, the secondary antibodies used have to be highly cross-adsorbed.
- IRDye® secondary antibodies have been completely optimized for fluorescent western blot, because they have been tested for over 600 different protein targets.
||Protein with higher expression (e.g. Housekeeping gene)
||Excitation: 679 nm
Emission: 696 nm
1:5,000 - 1:25,000
Protein lower expression
(Normally the protein of interest)
|Excitation: 778 nm
Emission: 795 nm
800 nm channel
1:5,000 - 1:25,000
Optimizing the Antibody
Get better signal-to-background ratio.
- In order to find the optimal antibody concentration, the primary and secondary antibodies have to be individually titrated. It is good to test various dilutions and choose the one that yields the highest signal-to-background ratio.
- Optimize conditions for individual primary and conjugated secondary antibody pairs separately. Only after that should multi-color analysis be attempted.
Steps and Reagents to Pay Attention to in the Protocol
- One of the issues with membranes is that they tend to autofluoresce in a high background. Therefore, when choosing a membrane, one has to make sure to choose specialist membranes that have been engineered to overcome this issue; such is the low fluorescence western membrane (PVDF).
- One of the undissolved particles within buffers that can settle in the membrane and create fluorescent artifacts is the milk powder in blocking buffer. Hence, high quality reagents are recommended in order to allow suitable time for all components to dissolve completely and filter sterilize all buffers.
- Membranes have to be handled with care. Blunt forceps are recommended to be used and scratching or creasing has to be avoided in order to prevent fluorescent artifacts.
- Membranes have to be marked with a pencil rather than a pen, because ink fluoresces.
- Bromophenol blue itself fluoresces - so either run the dye front off the gel, or cut the gel part off, which contains the dye, prior to transfer.
- The membrane has to be protected from light during incubation and steps have to be washed with aluminum foil.
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