ddH2O Plastic dish with lid Procedure – Probe: Prepare milk blocking solution by vortexing the following in a conical tube: TBST: 50 ml Milk block: 2.5 g Note: It may be useful to make up all block for procedure at this time, however, keep in mind that milk block spoils over time and spoiled block can alter results. ~ 100 mL TBST + Milk used for one membrane and nine independent antibody incubations Place block in plastic dish with lid, large enough to fit membrane. Turn power supply off, and disassemble sandwich to expose PVDF. Remove PVDF membrane from wattman paper with flat forceps. Quickly, rinse PVDF with ddH2O to remove excess methanol. Do not allow PVDF to dry out. Place PVDF immediately into Ponceau stain (see Ponceau protocol) or milk block, always keeping the transfer side up. Once in milk, snip membrane corner with razor blade to mark top from bottom, left from right. Block for 1 hour at room temperature. Leaving membrane in the dish, pour block off membrane, immediately replacing it with enough TBST to cover the entire membrane. Rinse on orbital for 10 minutes. Repeat 2 times. Mix enough block to cover entire membrane (usually ~15 ml needed depending on size of plastic) with desired concentration of primary antibody. Leaving the membrane in the dish, remove rinse from membrane, and add primary antibody mixture. Shake overnight on orbital at 4 degrees. Note: Milk will spoil overnight if not refrigerated, which may affect results. Wash membrane with TBST 3x for 10 minutes each. Mix enough block to cover entire membrane with desired concentration of secondary antibody. Remove rinse and incubate with secondary on orbital for 1 hour at room temperature. Rinse with TBST on orbital at room temperature 3x, for 10 minutes each. Notes: Never allow the membrane to dry out – have solutions ready so that when one comes off, the next can be immediately added. Be sure to keep stay oriented as to which side of the membrane is up and which is top versus bottom, or it can become very tricky to interpret results.
ddH2O Plastic dish with lid Procedure – Probe: Prepare milk blocking solution by vortexing the following in a conical tube: TBST: 50 ml Milk block: 2.5 g Note: It may be useful to make up all block for procedure at this time, however, keep in mind that milk block spoils over time and spoiled block can alter results. ~ 100 mL TBST + Milk used for one membrane and nine independent antibody incubations Place block in plastic dish with lid, large enough to fit membrane. Turn power supply off, and disassemble sandwich to expose PVDF. Remove PVDF membrane from wattman paper with flat forceps. Quickly, rinse PVDF with ddH2O to remove excess methanol. Do not allow PVDF to dry out. Place PVDF immediately into Ponceau stain (see Ponceau protocol) or milk block, always keeping the transfer side up. Once in milk, snip membrane corner with razor blade to mark top from bottom, left from right. Block for 1 hour at room temperature. Leaving membrane in the dish, pour block off membrane, immediately replacing it with enough TBST to cover the entire membrane. Rinse on orbital for 10 minutes. Repeat 2 times. Mix enough block to cover entire membrane (usually ~15 ml needed depending on size of plastic) with desired concentration of primary antibody. Leaving the membrane in the dish, remove rinse from membrane, and add primary antibody mixture. Shake overnight on orbital at 4 degrees. Note: Milk will spoil overnight if not refrigerated, which may affect results. Wash membrane with TBST 3x for 10 minutes each. Mix enough block to cover entire membrane with desired concentration of secondary antibody. Remove rinse and incubate with secondary on orbital for 1 hour at room temperature. Rinse with TBST on orbital at room temperature 3x, for 10 minutes each. Notes: Never allow the membrane to dry out – have solutions ready so that when one comes off, the next can be immediately added. Be sure to keep stay oriented as to which side of the membrane is up and which is top versus bottom, or it can become very tricky to interpret results.
Western%20blot%20protocol_Nystul.docx