Molecular Devices SpectraMax Paradigm User Manual page 59

Blocking Nonspecific Binding
To reduce noise, use blocking buffer to reduce non-specific protein from binding with the
membrane.
No single-blocking reagent is optimal for every antigen-antibody pair. Some primary
antibodies can exhibit greatly reduced signal or different nonspecific binding in different
blocking solutions. If you have difficulty detecting your target protein, changing the blocking
solution can dramatically improve the performance. If the primary antibody has worked well
in the past using chemiluminescent detection, try that same blocking solution for detection.
Other commonly used blocking buffers other than BSA are 3% casein and 5% non-fat milk.
Milk-based blockers can contain IgG that can crossreact with anti-goat antibodies. This can
significantly increase background and reduce antibody titer. Milk-based blockers can also
contain endogenous biotin or phosphoepitopes that can cause higher background.
To prevent background speckles on blots, use high-quality, ultra-pure water for buffers.
Do not over-block. Extended blocking times can cause loss of target protein from the
membrane.
Handling the Membrane
To scan the membrane in a SpectraMax Paradigm Multi-Mode Detection Platform, the
membrane must be placed in a Molecular Devices ScanLater™ Membrane Holder. See
Loading the Membrane into the Membrane Holder on page
Note: Handle membranes by their edges only, using clean forceps. Do not touch the
membrane with gloved or bare hands.
The maximum size for a membrane is 109 mm x 77 mm to let it fit in the membrane holder.
The Western Blot should be prepared using standard blotting procedures for the membrane
being used. For optimal results, use Millipore Immobilon FL (IPFL00010). If using PVDF, pre-
wet the membrane in 100% methanol.
Use enough antibody volume so that the entire membrane surface is sufficiently covered
with liquid at all times. Use heat-seal bags if the volume is limiting. Do not let an area of the
membrane dry out. Use agitation for all antibody incubations.
Small proteins can pass through the membrane during transfer ("blow-through"). To prevent
this, use a membrane with a smaller pore size or reduce the transfer time.
Allow the blot to dry for a minimum of 1 hour before detection.
Do not wrap the membrane in plastic when scanning.
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Chapter 2: Read Modes and Read Types
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