Drummond:Protein Slot Blot Protocol
If purified, suspend sample in PBS or TBS, or other appropriate buffer. Cell lysates or extracts also may be applied to the membrane or filter for detection of cellular proteins. If using a filtration manifold, dilute sample to 300-500 µl for a concentration of 1-10 µg/spot, then carry out serial dilutions of the sample to determine the optimal concentrations for subsequent binding assays. If not using a manifold, volumes of 2-5 µl should be used.
If using a filtration manifold to apply samples, float membrane in deionized water until completely wet, then soak in PBS or TBS until use. If spotting without a manifold, membrane should be left dry.
Apply sample to membrane directly with a micropipette, or use a filtration manifold. For purified samples, apply 1-10 µg/spot or well. Sample should be diluted in PBS, TBS or other buffer.
Multi-well filtration manifold Place 2 sheets filter paper, prewet in transfer buffer, on the filter support plate of the Manifold device. Place membrane on top of the filter paper and clamp the sample well plate into place. Apply low vacuum (enough to pull buffer through at approximately 1 mL/minute) and wash each well with 500 µL of sample buffer. While the vacuum is applied, add sample to the well and filter through. After sample has been applied to the membrane, wash with 500 µL of sample buffer. For best results, suspend sample in 300-500 µL of buffer. Unclamp and remove the sample well plate, taking care to leave the membrane on the filter paper. Remove the membrane from the Manifold device.
Direct sample application If a Manifold device is not used, apply sample to the membrane placed on top of 2 sheets of dry filter paper in 2-5 µL aliquots. If larger sample volumes are used, allow sample spot to dry prior to subsequent 2-5 µL aliquot addition. Note: If using a filtration manifold, low molecular weight samples should be filtered through slowly to prevent passage through the medium, or applied without vacuum.
Follow blocking and detection procedures as indicated for the NitroBind membrane