What gels and blots can and can’t tell you - interpreting (without overinterpreting) your results

[@thebumblingbiochemist]

So, in summary: gels can't tell you what is in a sample in ideal situations, it can tell you about relative sizes, purities, and quantities of molecules, but not their identity higher-up bands indicate bigger molecules* *molecular composition can impact how fast molecules run & thereforehow"big" they appear compared to the ladder more bands indicates lower purity** **you theoretically see everything that's there as separate bands, but the bands for similarly-sized molecules will overlap, and the quantities of some molecules might be too low to detect stronger bands indicates higher quantity*** ***the same concentration of smaller molecules has a lower quantity mass-wise and will bind less dye - so the same number of copies of smaller things have weaker signal than bigger things molecular composition also impacts dye binding and thus signal strength blots can't tell you what else is in a sample assuming you have good antibodies or probes, they can tell you whether a specific molecule is present**** but they can only tell you about the thing you're probing for- and nothing more! ****antibodies differ in strength so you can't directly compare band strength of different molecules - but you can compare strength of the same molecule probed for in different samples; also, antibodies can bind non-specifically to things, so include controls! similar holds true for nucleic acid probes - use longer probes and/or probes with higher Tms (stronger binding) for better specificity more on blots: blog form (text old, video & some graphics new) : bit.ly/blotcompass ; KZbin: kzbin.info/www/bejne/mqq6n56Yi66CqJY   more one western blots in particular: bit.ly/westernblotworkflow ; KZbin: kzbin.info/www/bejne/habRZ5Kra6pjb5I more on gel electrophoresis: kzbin.info/www/bejne/j6DMf5uuhbONgK8 & kzbin.info/www/bejne/j6DMf5uuhbONgK8 more on coomasssie staining: bit.ly/cbbgelstaining & kzbin.info/www/bejne/gnPWioaXj5ugeNE more on silver staining: bit.ly/silver_staining & kzbin.info/www/bejne/eF7IaJefmNyFqsU more on fluorescent nucleic acid stains: bit.ly/fluorescentstains & kzbin.info/www/bejne/j6DMf5uuhbONgK8 more on how DNA topology can affect how plasmids run: bit.ly/DNAtopology & kzbin.info/www/bejne/apWuYmaior-Xbs0                         more on protein purification: bit.ly/proteinpurificationtech more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@comicalkilla]

I just switched labs in May and am a 5th year in pharmacology. Your videos have been extremely helpful. You're an inspiration. Keep uploading! Stumbled upon your website and now your KZbin videos are part of my morning coffee ☕ ritual

[@thebumblingbiochemist]

Thanks so much! I'm so happy I could help - best of luck with your studies!

[@MarianaAguilarMorales]

great video! thank you so much for selflessly sharing all you knowledge!

[@thebumblingbiochemist]

Glad I could help!

[@ziyatufail]

can we load our loading protein control in lesser concentration than our protein of interest in another gel???

[@thebumblingbiochemist]

I don't understand your question sorry!

[@ziyatufail]

@@thebumblingbiochemist lets say i want to check the expression of lc3 protein , so according to literature/guidelines i have to load atleast 20ug of protein per well but for normalisation i have to reprobe the same blot with Actin antibody. But herez the thing 20ug is too much for actin , i think 4ug for actin is enough for crisp bands. So can i normalise my protein of interest LC3 (20ug) with actn (3ug) from the same set of cell lysates (samples)????

[@thebumblingbiochemist]

They wouldn't be in the same loading anymore if you did that, which would thus not allow you to use it as a loading control