Expression and purification of His-tagged proteins from E. coli
Жүктеу.....
Пікірлер: 66
@yolisamagibile9 ай бұрын
All Biochemistry Master's Students looking to understand the method of protein expression for your project hands up ✋🏽
@jonathanndunguru1195
5 ай бұрын
🙌
@Jenny-ym5rg
19 күн бұрын
holler 🖐
@mna1592 жыл бұрын
Please continue this, it's definitely a channel worth subscribing
@katerynakozyrieva5631 Жыл бұрын
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
@michaeloseiappiah70033 жыл бұрын
Good presentation with detailed explanation
@sahilseikh9902 Жыл бұрын
I'm glad that I found your channel. It's really a nice and detailed Protein extraction video on KZread that's really helpful for beginners like me 😃
@lulub50592 жыл бұрын
Great video! Great technique. Thank you.
@zodeirefo22212 жыл бұрын
Thank you so much for this!
@TheSergeyVlasenko Жыл бұрын
Great performance, great explanation. Thank you.
@aisyahmoktarroji1673 жыл бұрын
Thanks a lot!! Very good explanation
@hesnayigit88402 жыл бұрын
Thank you very much, this is great for teaching with limited lab.
@Chickynugget22 жыл бұрын
Hi, this video was great!! Maybe you can do additional videos on SDS-page interpretation (maybe with different proteins and conditions).
@aleksandar20462 жыл бұрын
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 😂💪🏼
@user-zd7ns9ij5g
Жыл бұрын
Your whole MS thesis was on recombinant protein production?
@aleksandar2046
Жыл бұрын
@@user-zd7ns9ij5g 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
10 ай бұрын
not really, some proteins are difficult to purify, talking from experience, especially uncharacterized proteins. @@aleksandar2046
@panoskre Жыл бұрын
So helpful, thanks a lot!!
@kristoffersoelmark6742 жыл бұрын
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
@leahmwendwa5638 ай бұрын
Thanks for the video. As a first-year PhD student, I needed this video to get my feet on the ground. Thanks a lot
@user-id9lf2pi4n3 жыл бұрын
Thanks a lot!!
@tinasheprincemaviza752 жыл бұрын
I liked the cotton idea :) on gel staining part of the protocol. Useful indeed
@blanket6863 Жыл бұрын
love this video thank you!!
@hitkarshkushwaha24342 жыл бұрын
Outstanding sir
@guleena7852 жыл бұрын
Really good and well explained 👍
@jenifermunozgomez21023 ай бұрын
Thank you !!!
@lmtrevino72 жыл бұрын
thank you
@benysmart16432 жыл бұрын
Thank you
@adronung18922 жыл бұрын
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
2 жыл бұрын
Use a 5 mL HiTrap with an FPLC system
@elijahfletcher5944 Жыл бұрын
Quality content
@falalalalamyohmy2 жыл бұрын
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!
@spectator592 жыл бұрын
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?
@Hoxgene3 жыл бұрын
very nice
@lucisleesion88243 жыл бұрын
Hello protein purifiers, hahahaha
@shawnbai95432 жыл бұрын
I want to see how the running gel looks like.
@dr.agupta Жыл бұрын
It is always better to add lysozyme AFTER resuspending the pellet.
@MohammedAli-bj9jk2 жыл бұрын
whats the name of the spectrophotometer machine you were using?
@ambreenkanwal89792 жыл бұрын
Hi Great Job Can I have this protocol in written form So i cannot miss any point. It would be highly appreciated
@yordanostselasi45502 жыл бұрын
Do you have a protocol please
@spacescience1002 жыл бұрын
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?
@amitmaurya2792 жыл бұрын
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
@atpsynthase17983 ай бұрын
Can you share some references you use to do in this video? Thank you so much
@harshitasharma795410 ай бұрын
Hi where is your lab I have a few questions
@sjoerdfennema984 Жыл бұрын
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.
@Lussid Жыл бұрын
10:40 for Day 4
@suraalbermani6212 жыл бұрын
please write the name of manufacture Ni-nickel resin
@inastasia48712 жыл бұрын
May I know what does it mean by to wash with 10 column volume? I encounter this in an article
@kwanlab4034
2 жыл бұрын
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.
@jinty12322 жыл бұрын
LB should be pH'd to 7.
@TheSakapi3 жыл бұрын
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
2 жыл бұрын
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
2 жыл бұрын
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.
@mudondojoyce30902 жыл бұрын
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
2 жыл бұрын
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
@soumendash39113 жыл бұрын
Where is the lab situated?
@kwanlab4034
2 жыл бұрын
We're in Montreal
@rongyinghuang6033 жыл бұрын
what dose the energy you use for sonicator the bacteria?
@kwanlab4034
2 жыл бұрын
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)
@lucisleesion88243 жыл бұрын
how do you bring the filming apparatus into the lab? With parafilm covered?
@kwanlab4034
2 жыл бұрын
It's just a cell phone camera and a tripod.
@Stronger.1192 жыл бұрын
Is the ice important and why?
@user-dr9jh6rp6y
Жыл бұрын
To avoid protein degradation
@arosas19913 жыл бұрын
Is this how you can make human growth hormone?
@kwanlab4034
2 жыл бұрын
Maybe this reference helps: Olson, K.C. et al. (1981) Nature, 293, 408-411 doi.org/10.1038/293408a0
@MrEvertonian20
Жыл бұрын
@@kwanlab4034 ? Link doesn’t work. How to make HGH?
Пікірлер: 66
All Biochemistry Master's Students looking to understand the method of protein expression for your project hands up ✋🏽
@jonathanndunguru1195
5 ай бұрын
🙌
@Jenny-ym5rg
19 күн бұрын
holler 🖐
Please continue this, it's definitely a channel worth subscribing
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
Good presentation with detailed explanation
I'm glad that I found your channel. It's really a nice and detailed Protein extraction video on KZread that's really helpful for beginners like me 😃
Great video! Great technique. Thank you.
Thank you so much for this!
Great performance, great explanation. Thank you.
Thanks a lot!! Very good explanation
Thank you very much, this is great for teaching with limited lab.
Hi, this video was great!! Maybe you can do additional videos on SDS-page interpretation (maybe with different proteins and conditions).
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 😂💪🏼
@user-zd7ns9ij5g
Жыл бұрын
Your whole MS thesis was on recombinant protein production?
@aleksandar2046
Жыл бұрын
@@user-zd7ns9ij5g 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
10 ай бұрын
not really, some proteins are difficult to purify, talking from experience, especially uncharacterized proteins. @@aleksandar2046
So helpful, thanks a lot!!
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
Thanks for the video. As a first-year PhD student, I needed this video to get my feet on the ground. Thanks a lot
Thanks a lot!!
I liked the cotton idea :) on gel staining part of the protocol. Useful indeed
love this video thank you!!
Outstanding sir
Really good and well explained 👍
Thank you !!!
thank you
Thank you
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
2 жыл бұрын
Use a 5 mL HiTrap with an FPLC system
Quality content
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!
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?
very nice
Hello protein purifiers, hahahaha
I want to see how the running gel looks like.
It is always better to add lysozyme AFTER resuspending the pellet.
whats the name of the spectrophotometer machine you were using?
Hi Great Job Can I have this protocol in written form So i cannot miss any point. It would be highly appreciated
Do you have a protocol please
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?
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
Can you share some references you use to do in this video? Thank you so much
Hi where is your lab I have a few questions
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.
10:40 for Day 4
please write the name of manufacture Ni-nickel resin
May I know what does it mean by to wash with 10 column volume? I encounter this in an article
@kwanlab4034
2 жыл бұрын
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.
LB should be pH'd to 7.
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
2 жыл бұрын
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
2 жыл бұрын
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.
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
2 жыл бұрын
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
Where is the lab situated?
@kwanlab4034
2 жыл бұрын
We're in Montreal
what dose the energy you use for sonicator the bacteria?
@kwanlab4034
2 жыл бұрын
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)
how do you bring the filming apparatus into the lab? With parafilm covered?
@kwanlab4034
2 жыл бұрын
It's just a cell phone camera and a tripod.
Is the ice important and why?
@user-dr9jh6rp6y
Жыл бұрын
To avoid protein degradation
Is this how you can make human growth hormone?
@kwanlab4034
2 жыл бұрын
Maybe this reference helps: Olson, K.C. et al. (1981) Nature, 293, 408-411 doi.org/10.1038/293408a0
@MrEvertonian20
Жыл бұрын
@@kwanlab4034 ? Link doesn’t work. How to make HGH?