SDS-PAGE calculations - walk-through of how to calculate and prepare to load equal masses per lane
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- Опубликовано: 2 окт 2024
- Sometimes, you want to calculate to load equal total protein amounts in different lanes of an SDS-PAGE gel. This allows you to do things like compare purity of different protein preps or compare expression of a protein in different cellular conditions with a western blot. I made this video to help one of my students with some real-world calculations we’re doing, so thought I'd share it in case it could be helpful to others as well. Apologies for sloppiness - just started trying out using an Apple pencil and iPad with Goodnotes for handwriting and am obviously not very good at it!
The overall strategy is
measure sample concentrations
decide what mass and volume to load (typicallly 2-4 ug of a purified protein (for coomassie, less for western) or 20-50 ug of a lysate)
dilute all samples to the concentration needed to load that much in that volume (taking into account the volume and dilution from the sample-loading buffer) - make more than you need
mix with the sample loading buffer
load and go!
summary/review starts at 36:11
It should say 10µL dye at 55 - sorry!
slides: drive.google.c...
more on SDS-PAGE: bit.ly/sdspager...
more on metric conversions: blog: bit.ly/metric_... ; RUclips: • Metric conversions & s...
more on sig figs (which I ignored in this video) here: bit.ly/signific... & • Figuring out sig figs ...
more on dimensional analysis: bit.ly/dimensio... & • Dimensional analysis (...
more about concentrations: bit.ly/sciences... ; RUclips: • Concentration - ways t... & • Working with relative ...
and a couple cheat sheets for molecular weights: drive.google.c...
and concentrations: drive.google.c...
more lab-mathy things: thebumblingbio... & • lab math-y stuff
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbioc...
thank you so much for this video, this is exactly what I needed. I'm a noob to biochem labs and western blot, I was very confused with the calculations and maybe its so basic that noone seem to be able to explain to me, you saved my life! cant wait to learn from you other videos!
Glad it was helpful!
Love your vids. When I started in my lab, your channel was my go to for westerns!!! I would love to see you do a qPCR video
Thank you! I have done several on qPCR including: ruclips.net/video/4WPkAMB_dPU/видео.html & ruclips.net/video/kp4ZX2lOr6w/видео.html
Thank you for your time and effort!
& On ecoli i used to have no insoluble pellet after boling but now I'm getting more & more insoluble pellet after boling ! Any mistake from my side!
Don't know sorry - is the buffer the same? SDS can precipitate out with potassium
55:16 should we add 10uls of sample buffer instead of 6uls, considering sample buffer is 1/4th of total volume. Thanks
yes - typo sorry! thanks for the catch!
I am 4th year UG, not a great student not bad either but these calculations are still nightmare and confusing for me(not because I am bad at math I even took advanced calculus clasesses) but this video helped!
So great to hear that this was actually helpful!
Haha if only you had uploaded this 7 months ago! My lab classmates and I were struggling on these calculations but better late than never! Thank you for your excellent explanation :)
Glad you think they would have helped at least!
Super helpful and well explained ! I was stock on the calculations and now everything is clear. Thanks !
Glad it helped!