Expression and purification of His-tagged proteins from E. coli

[@yolisamagibile]

All Biochemistry Master's Students looking to understand the method of protein expression for your project hands up ✋🏽

[@jonathanndunguru1195]

🙌

[@Jenny-ym5rg]

holler 🖐

[@mna159]

Please continue this, it's definitely a channel worth subscribing

[@katerynakozyrieva5631]

first of all - thanks, it really helped me to understand several steps of this procedure better! secondly, it's the best unintentional asmr i've heard in my entire life, please, continue

[@michaeloseiappiah7003]

Good presentation with detailed explanation

[@aleksandar2046]

Ahh this was such a joy to watch. I am currently doing my master's thesis on recombinant production of some protein and watching you do this whole process was such a cool experience 😂 I am so motivated now for my GST affinity purification tomorrow 😂💪🏼

[@F.F-p4m]

Your whole MS thesis was on recombinant protein production?

[@aleksandar2046]

@@F.F-p4m and its characterization and potential application in serological test development. But yes, the central part of it was recombinant production of proteins. Sounds pretty underwhelming, right?

[@thaborolffy7721]

not really, some proteins are difficult to purify, talking from experience, especially uncharacterized proteins. @@aleksandar2046

[@ambreenkanwal8979]

Hi Great Job Can I have this protocol in written form So i cannot miss any point. It would be highly appreciated

[@leahmwendwa563]

Thanks for the video. As a first-year PhD student, I needed this video to get my feet on the ground. Thanks a lot

[@Chickynugget2]

Hi, this video was great!! Maybe you can do additional videos on SDS-page interpretation (maybe with different proteins and conditions).

[@sahilseikh9902]

I'm glad that I found your channel. It's really a nice and detailed Protein extraction video on KZbin that's really helpful for beginners like me 😃

[@falalalalamyohmy]

Hello! My name is Maria and I am a PhD student working on a project that works to collate various biological techniques for early career researchers in the lab. I really loved your tutorial video for protein purification, and was wondering whether we could get in touch to discuss it further and other similar protocols. Please let me know I'd love to hear back from you!

[@tinasheprincemaviza75]

I liked the cotton idea :) on gel staining part of the protocol. Useful indeed

[@lucisleesion8824]

Hello protein purifiers, hahahaha

[@adronung1892]

I do protein expression with 6 liters of culture. After centrifugation of the lysate, the supernatant is passed onto the Ni-NTA agarose column. It takes several days for the supernatant from such a large culture to pass through the column or immediately by syphoning the supernatant out of the column by applying a vacuum to the base of the column.

[@C-Wam]

Use a 5 mL HiTrap with an FPLC system

[@randomaccmeme]

Awesome video, but it's criminal that you didn't show how the gel looks like after coomassie destaining

[@kristoffersoelmark674]

Aabsolute master! Thanks my dude - this helped a lot! For the viewer's sake you might include on-screen stats for the reagents used. Thanks again! :D

[@TheSergeyVlasenko]

Great performance, great explanation. Thank you.

[@adarshguptak]

It is always better to add lysozyme AFTER resuspending the pellet.

[@aisyahmoktarroji167]

Thanks a lot!! Very good explanation

[@lulub5059]

Great video! Great technique. Thank you.

[@shawnbai9543]

I want to see how the running gel looks like.

[@spectator59]

I appreciated the level of detail you described here, thanks. Using your technique, about how long does it take you to grow and purify a 1L culture?

[@zodeirefo2221]

Thank you so much for this!

[@spacescience100]

How do you determine what gradient of SDS-PAGE gel to use since there are multiple products on the market that range from 4%-12, or 4%-20% gradient?

[@hesnayigit8840]

Thank you very much, this is great for teaching with limited lab.

[@guleena785]

Really good and well explained 👍

[@panoskre]

So helpful, thanks a lot!!

[@hitkarshkushwaha2434]

Outstanding sir

[@atpsynthase1798]

Can you share some references you use to do in this video? Thank you so much

[@АртурАхтямов-я8т]

Thanks a lot!!

[@blanket6863]

love this video thank you!!

[@jinty1232]

LB should be pH'd to 7.

[@elijahfletcher5944]

Quality content

[@jenifermunozgomez2102]

Thank you !!!

[@avishekshaw3709]

Thanks a lot :)

[@lmtrevino7]

thank you

[@Hoxgene]

very nice

[@sjoerdfennema984]

Please tell me how you made your elution buffer, everytime I add imidazole the pH rises above whats needed. And adjusting it with acid is not possible because of possible interference.

[@harshitasharma7954]

Hi where is your lab I have a few questions

[@khushigoel052]

U have used dnase before sonication.. does the addition of dnase before sonication impacts lysis differently or it does not affect the lysis

[@gulafshahussain6113]

Can we resuse this columns.. Or we need to add agarose colum each time?

[@YirgalemHabte1966]

Do you have a protocol please

[@amitmaurya279]

What is the success/trial ratio of this process, if i follow the process exactly, will i be able to express the protein or does it takes few trials

[@MohammedAli-bj9jk]

whats the name of the spectrophotometer machine you were using?

[@suraalbermani621]

please write the name of manufacture Ni-nickel resin

[@khushigoel052]

Which protein you are expressing and purifying

[@TheSakapi]

very good presentation well done,i have a question if you don't mind i which way should i adjust ph? my protein has PI=4,3 and i'm really confused if i have to regulate TRIS buffer ph to 6

[@kwanlab4034]

The pH of the buffer depends on your protein, but for binding to Ni-NTA it needs to be between 7.5 and 9.

[@adronung1892]

The isoelectric point (pI) of your protein is the pH at which your protein is least soluble. If you want your protein to be soluble, your protein pH should be distance from the pI. Tris does not buffer to pH 6 because the pKa of Tris is 8.1 and pH 6 is two pH units away from the pKa of the buffer. Low pH elutes proteins from Ni-NTA Agarose, because it protonates the histidine residues in the polyhistidine tag and thus your recombinant target protein cannot bind to the resin.

[@inastasia4871]

May I know what does it mean by to wash with 10 column volume? I encounter this in an article

[@kwanlab4034]

By "column volume" I'm referring to the volume of resin inside the column, so if there is 1 mL of resin, 10 column volumes is 10 mL.

[@Lussid]

10:40 for Day 4

[@mudondojoyce3090]

thank you the videos ,i am doing sds page but my run wont start even after adding running buffer to the mark .i use the same mini gel tank like yours ,though it leaks ,could it be the issue ?help me from this confusion please

[@C-Wam]

Use fresh running buffer, ensure you have the electrodes the correct way, ensure no leaks, make sure gel is entered the right way, remove the white tape at the bottom of the pre-case gel if you are using those

[@arosas1991]

Is this how you can make human growth hormone?

[@kwanlab4034]

Maybe this reference helps: Olson, K.C. et al. (1981) Nature, 293, 408-411 doi.org/10.1038/293408a0

[@MrEvertonian20]

@@kwanlab4034 ? Link doesn’t work. How to make HGH?

[@soumendash3911]

Where is the lab situated?

[@kwanlab4034]

We're in Montreal

[@rongyinghuang603]

what dose the energy you use for sonicator the bacteria?

[@kwanlab4034]

We use a Fisherbrand™ Model 505 Sonic Dismembrator (500W) set to 25% amplitude. (www.fishersci.ca/shop/products/fisher-scientific-model-505-sonic-dismembrator-4/p-3974677)

[@Stronger.119]

Is the ice important and why?

[@КекКеков-ф1с]

To avoid protein degradation

[@lucisleesion8824]

how do you bring the filming apparatus into the lab? With parafilm covered?

[@kwanlab4034]

It's just a cell phone camera and a tripod.

[@benysmart1643]

Thank you