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Nice video overall, just a few notes: 100 degree Boiling for 15 minutes is ill advised and will lead to protein hydrolysis for many proteins. 95C for 5-10 minutes, or 70C for 10-20 minutes is more than plenty for 99% of western blotting applications. 150-200V is fine with Invitrogen's Bis-Tris Gels, especially if you use cold Running Buffer to start. For Invitrogen Dry Transfer pre-assembled sandwich, they SPECIFICALLY advise to not activate the PVDF membrane as it is already pre-activated. Washing with Blocking Buffer is only necessary when using blocking buffers that are resuspended just prior to using. This is to get rid of any particulates. With commercial blocking buffers, or stock solutions of blocking buffers that are already in solution, you can proceed immediately to adding the primary antibody after the blocking step. Fluorescent labeled primary antibodies are quite nice as well. lastly and most importantly: adding the HRP substrate onto the membrane while shaking it leads to much more consistent detection rather than placing the membrane on top of a puddle of the reagent...This won't disperse as evenly and there's a decent chance it won't cover the membrane evenly. The sheet protector is a nice thing to have as it'll keep your blot from drying out during long exposures.
Addgene does not regularly monitor comments posted here, so we may not see your question immediately. We’d be happy to answer any questions sent to help@addgene.org as soon as possible. Please include the name of the video along with any questions so our support team can help. Thanks!
Nice video overall, just a few notes:
100 degree Boiling for 15 minutes is ill advised and will lead to protein hydrolysis for many proteins.
95C for 5-10 minutes, or 70C for 10-20 minutes is more than plenty for 99% of western blotting applications.
150-200V is fine with Invitrogen's Bis-Tris Gels, especially if you use cold Running Buffer to start.
For Invitrogen Dry Transfer pre-assembled sandwich, they SPECIFICALLY advise to not activate the PVDF membrane as it is already pre-activated.
Washing with Blocking Buffer is only necessary when using blocking buffers that are resuspended just prior to using. This is to get rid of any particulates. With commercial blocking buffers, or stock solutions of blocking buffers that are already in solution, you can proceed immediately to adding the primary antibody after the blocking step.
Fluorescent labeled primary antibodies are quite nice as well.
lastly and most importantly: adding the HRP substrate onto the membrane while shaking it leads to much more consistent detection rather than placing the membrane on top of a puddle of the reagent...This won't disperse as evenly and there's a decent chance it won't cover the membrane evenly.
The sheet protector is a nice thing to have as it'll keep your blot from drying out during long exposures.
thanks for the video!