Western Blot Protocol

Looking for a way to visualize a protein from cells or tissue samples? Maybe using... the dreaded Western Blot? Well fear not! Meghan from Addgene is here to make sure you'll be running, transferring, and chemiluminescing like a pro by the time you finish this video.

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00:00 - Intro
00:47 - Materials
02:00 - Preparing Samples
03:57 - Preparing the Gel
07:21 - Using the Transfer Apparatus
10:20 - Primary Antibody Incubation
12:09 - Secondary Antibody Incubation
13:00 - Prepare the Detection Reagent
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Credits:
Featuring: Meghan Rego
Written By: Meghan Rego, Rachel Leeson, & Quintin Marcelino
Directed, Animated, & Edited by: Quintin Marcelino
Videography by: Quintin Marcelino
Sound Design by: Quintin Marcelino
Designs by: Jason Snair
Addition Designs by: Quintin Marcelino
Music by: Anno Domini Beats & Bail Bonds, courtesy Youtube Audio Library.

Disclaimer: The SOP presented here has been designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene for informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with Addgene, can be held responsible for your use of the information contained in or linked to from these web pages.

[bytesizebiotech]

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.