SDS-PAGE calculations - walk-through of how to calculate and prepare to load equal masses per lane

Описание: 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: https://drive.google.com/file/d/1Ugnj...

more on SDS-PAGE: http://bit.ly/sdspageruler

more on metric conversions: blog: https://bit.ly/metric_calcs ; YouTube:    • Metric conversions & scientific notation i...    

more on sig figs (which I ignored in this video) here: http://bit.ly/significantfiguress &    • Figuring out sig figs - working with signi...    
 
more on dimensional analysis: http://bit.ly/dimensionalanalysising &    • Dimensional analysis (aka factor-label met...   
 
more about concentrations: http://bit.ly/sciencestocksolutions ; YouTube:    • Concentration - ways to report it (molarit...   &    • Working with relative concentrations (in t...    
 
and a couple cheat sheets for molecular weights: https://drive.google.com/file/d/1TqCR...   
 
and concentrations: https://drive.google.com/file/d/1v7pf...    

more lab-mathy things: https://thebumblingbiochemist.com/let... &    • lab math-y stuff  

more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com