Size Exclusion Chromatography (aka Gel Filtration): theory & practice of preparative & analytical

[@marksteve8373]

Thank God I discovered your channel! You are amazing, Brianne! I'm a cellular molecular biology undergrad and was kind of struggling with the whole process of using the chromatography machine. We have NGS one, so I guess it looks a little different from yours but works the same. I haven't watched all of your videos yet, but if you haven't made it already, it would really be great if you could make a video doing a protein purification and explaining the reasoning behind each step as you do it. Thank you again & I'm really inspired by you!

[@thebumblingbiochemist]

Thanks so much! I'm really happy I could help. I have a page of posts on my blog discussing protein purification you might find helpful: bit.ly/proteinpurificationtech Here's a video on using an AKTA: bit.ly/aktainaction And one on the protein purification workflow: kzbin.info/www/bejne/i2a1aKeVdr6indk I also have a playlist on protein purification. Hope something there can help!

[@beepboopbs98]

performed GFC for the first time this year but we used sephadex G-50 also we don't have an automated column or digital detector so we packed our own column with gel and poured the buffer and eluted the sample in small test tubes and took their absorbance readings one by one on the spectrophotometer (we had around 40 labelled fractions!) we even plotted the peaks on a graph paper and everything . (idk why am i telling u all of this great vid btw thanks for ur insights)

[@thebumblingbiochemist]

Oh wow! That's quite the effort you must have put in. Hope it was successful! (and thank you!)

[@MrAlperne]

love to watch your videos! It is very educational for me to watch :)

[@thebumblingbiochemist]

Great to hear - thank you!

[@TheCD45]

Really great vlog! Our lab is just thinking of setting up a low-pressure chromatography set-up LC set-up but is sad that the system from bio-rad has been discontinued. Can you recommend a good system for us to consider? I have a beginner's knowledge of the equipment and am scratching my head with lots of options, my goal is to do SEC from my cell culture supernatant and other is to enrich proteins for glycosylation and perform purification.

[@thebumblingbiochemist]

Thank you so much! Maybe the AKTA Start system?

[@bitbybit5770]

Hey, Brianne. Great video. May I know how do you make all these infographs/figures?

[@thebumblingbiochemist]

Thank you! I use Adobe Illustrator and I have some videos on it.

[@stephlemus9448]

Hi, Thank you for the video. I have a question, I am having problems with the purification, I am purifying a protein doing IMAC and then Amilose, and at the end SEC, my PI suggested that I may not inject the sample at the right time; for SEC, what is the optimal time to inject the sample?

[@thebumblingbiochemist]

You can inject it into the loop "whenever", but wait until the column is fully equilibrated before putting it on the column

[@madhurik8389]

Great video.. Could you please male video on nucleic acid Binding protein purification. How do you manage to get it RNA free. As I see it on Twitter.

[@thebumblingbiochemist]

Thanks! It depends on the protein. In my case the protein binds very strongly to endogenous RNAs, so I separate the RNA-bound from the endogenously-free with cation exchange chromatography. If the binding is weaker, you can try using high salt to get the RNA un-stuck

[@samanthahilston2241]

How many fold does your sample get diluted during a sec run with the sec columns you use most? Also, does the subsequent concentration step lead to just as much or less aggregation of your protein compared to what you were able to separate out during your first sec run? Love you videos! Thanks!

[@thebumblingbiochemist]

Thanks! Dilution before/through will depend on the sample volume and loop volume and column packing and stuff. Not really sure. Otherwise "dilution" can actually just be proteins separating. I typically inject ~500uL and concentrate ~2.5mL. I don't usually get precipitation during the subsequent concentration. Hope that helps

[@soulsymphony5111]

size exclusion chromatography using a syringe rather than FPLC .. would you happen to know how that works ?

[@thebumblingbiochemist]

The syringe is just to inject it into the FPLC