![]() *Make sure to select appropriate secondary antibody depending on the primary antibody’s host species. Incubate membrane in HRP conjugated secondary antibody at a dilution of 1:10,000-1:30,000 in 1% NFDM in 1X TTBS while rocking for 1 hour at room temperature.Discard primary antibody solution and wash membrane using 1X TTBS 3 x 5 minutes at room temperature.Incubate membrane in primary antibody overnight while rocking at 4˚C.If experiencing significant background noise, try using 1% BSA in 1X TTBS to dilute primary antibody.Optimal primary antibody dilutions should be determined experimentally using a dilution curve.Using the manufacturer’s recommended dilution, dilute primary antibody in 1% NFDM in 1X TTBS, making sure final volume will completely cover membrane during incubation.If experiencing significant background noise, try using 3% BSA in 1X TTBS as the blocking buffer.>.Block membrane in 5% nonfat dried milk (NFDM) in 1X TTBS for 30 minutes while rocking at room temperature to saturate any free binding sites on the membrane.If using PVDF, membrane must be activated by immersing it in methanol for 30 seconds.If using a large trough gel for a single lysate, cut and label individual strips. Cut and label desired number lanes from membrane.For long term storage, seal protein blot in a plastic bag and store in dark or dimly lit area. The markers will fade over time or wash away in any buffer. Mark molecular weight markers and lanes on the membrane using a gel pen.Ponceau S staining is reversible and will not interfere with antibody labeling of the membrane. Collect excess Ponceau S for reuse and make sure to thoroughly rinse membranes in dH 2O after staining to remove any remaining excess Ponceau S on the membrane and so protein bands are visible. Stain your membrane with Ponceau S after transfer to confirm successful protein transfer and to determine the exact alignment of lanes and placement of the proteins on the gel.A Kimwipe can be placed in the corner of the box to speed up the destaining process. Discard Coomassie Blue and soak in gel destain overnight rocking at room temperature. After transfer, rinse gel with dH 2O and incubate gel in Coomassie Blue to verify transfer efficiency.After transfer, rinse membrane thoroughly in dH 2O to remove any remaining transfer buffer on the membrane and air dry the membrane until completely deactivated (solid white).Adjust transfer times based on manufacturer’s recommendations and place an additional membrane in transfer to collect any protein that has potentially blown through the primary membrane. Larger molecular weight (~150-250 kDa) proteins will take a longer time to transfer than smaller molecular weight (~25-100 kDa) proteins.Set the voltage, amperage, and time based on manufacturer’s recommendations.Carefully remove any air bubbles present between the gel and the membrane with a roller, as bubbles will prevent proteins from transferring from the gel to the membrane.If gel front begins to exhibit a “smile”, reduce voltage.A 7.5% gel will run the fastest, a 15% the slowest. The run time for the gel will vary depending on the percentage of the gel. Allow gel to run until the dye front has passed through the gel.Run gels per electrophoresis apparatus’ manufacturer’s recommendations.Molecular weight ladder also must be brought up to same volume with the ‘blank buffer’ in multi-lane gels. In any empty lanes, load a ‘blank buffer’ consisting of lysis buffer containing 4X sample buffer at the same volume as samples. When using multi-lane gels, load equal volumes of sample to each lane to prevent lateral band spreading.For trough gel load ~0.5-1.0 mg of protein. For a multi-lane gel load ~5-50 µg of protein per lane. Using gel loading pipette tips, load cooled samples and protein marker onto gel.Assemble gels in the electrophoresis apparatus and add 1X running buffer to recommended fill level.When testing multiple antibodies using the same lysate, try using a stacking layer with one large trough instead of multiple lanes to maximize the number of strips available for testing.Add TEMED and 10% APS as last ingredients of gel recipe since they are responsible for starting the polymerization process.(Gel recipes and recommendations are listed below). Prepare appropriate percentage SDS gel based on the molecular weight of the protein of interest.
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