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General Hints and Tips, chemiluminescent detection
Optimization of the primary and secondary antibodies are recommended for best performance (see Invest time in antibody optimization)
During immunodetection, a sufficient volume of solution should be added to adequately cover the membrane. Containers should be agitated gently on a mixer platform. If more than one blot is placed in a container, insufficient buffer volume will cause the blots to stick together.
When washing, the volume of wash buffer should be as large as possible. Brief rinses of the membrane in wash buffer before incubation will improve washing efficiency.
Adding 0.5M NaCl and 0.2% SDS to the wash buffer (high salt buffers, washing for 30 min, followed by distilled water) after secondary antibody incubation can sometimes considerably reduce the background on PVDF-membranes. This is especially recommended for high abundant proteins.
Do not use Sodium Azide as a preservative for buffers to be used in immunodetection as it is an inhibitor of Horseradish Peroxidase (HRP).
Phosphatases in the blocking solution may dephosphorylate blotted proteins.
Optimum Tween 20 concentrations will vary to suit specific experiments, but 0.1% (v/v) Tween 20 is suitable for most blotting applications.
Dry milk powder cannot be used with biotin-avidin systems.
For minimising uneven staining of the detection reagent, which may result in uneven staining pattern on the film, first mix the detection solutions A+B and pour it into a clean container, then place the probed membrane in the detection solution and agitate gently the membrane to cover the entire surface (1 min for Amersham ECL™, 5 min for ECL Plus™ , and 5 min for ECL Advance™).
The protein blots can either be placed with the protein side down on a fresh piece of SaranWrap™ or in an A4 plastic file folder (which is more convenient), before it is placed in the X-ray film cassette. Make sure to gently remove any air bubbles after wrapping.
Although all the different working mixes of the Amersham ECL, ECL Plus and ECL Advance reagents are stable for longer time periods, it is recommended that reagents are mixed immediately before use. In the event that mixed reagents need to be left before use, protect from light by wrapping the container in foil or by storing in the dark. For reproducible performance equilibrate to room temperature before use.
Do not allow the membrane to dry out at any time during the immunodetection procedure.
If exposure times of less than 5 seconds are routinely required, it is recommended that the antibodies used are further diluted as it is difficult to perform such short exposures.
Ensure there is no free detection reagent in the cassette; the film must not get wet.
Fading signal can occur with many chemiluminescent substrates. The reason for this phenomena is unknown. The following can be tried out:
- ensure that the detection reagents have not yet expired and have been stored properly; the solutions shall not be kept in room temperature for too long or be exposed to direct light
- make sure that the caps on the detection solution flasks have not been mixed, which can contaminate the solutions.
- do not contaminate the solutions with the same pipette tips.
High overall background can be minimized by higher dilution of the HRP-conjugated secondary antibody.
High non specific signal can be elevated by higher dilution of primary antibody or reduced protein load on the gel.
Non specific signal can be reduced by diluting the primary and secondary antibodies in the blocking solution.
If the membrane should be stripped before another antibody probing, the blot should never allow to dry between rounds of immunodetection. Any residual molecules will bind permanently to the membrane if it is allowed to dry.
For stripping protocol (see different Amersham ECL detection protocols)
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