![]() If larger sample volumes are used, allow sample spot to dry prior to subsequent 2-5 µL aliquot addition. 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. Remove the membrane from the Manifold device. Unclamp and remove the sample well plate, taking care to leave the membrane on the filter paper. For best results, suspend sample in 300-500 µL of buffer. After sample has been applied to the membrane, wash with 500 µL of sample buffer. While the vacuum is applied, add sample to the well and filter through. Apply low vacuum (enough to pull buffer through at approximately 1 mL/minute) and wash each well with 500 µL of sample buffer. Place membrane on top of the filter paper and clamp the sample well plate into place. Place 2 sheets filter paper, prewet in transfer buffer, on the filter support plate of the Manifold device. Sample should be diluted in PBS, TBS or other buffer. For purified samples, apply 1-10 µg/spot or well. If spotting without a manifold, membrane should be left dry.Īpply sample to membrane directly with a micropipette, or use a filtration manifold. 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 not using a manifold, volumes of 2-5 µl should be used. 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. Cell lysates or extracts also may be applied to the membrane or filter for detection of cellular proteins. If purified, suspend sample in PBS or TBS, or other appropriate buffer. ![]()
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