Thank you you explained that 2 substances are in ion column...subs A directly attached to resin and subs B that is bound to it by an ionic bond and that B is the one to be exchanged with the analyte...thank you for revealing this
if the ph of the medium is lower than the pI of the protein, then the protein will be protonated and hence will be positively charged. If the ph is higher than pI then less proton are available in the medium and so the protein will lose its hydrogens from the side chains and become negatively charged.
Another rule, is that you should use a pH at least 1 unit away from the PI in order to make sure the protein/peptide is completely protonated/deprotonated. So, if the PI is 2, and the pH is 2.3, you will probably not get good retention of your target peptide.
Thank you for these great videos! My biotech LAB classes are only virtual right now (which is stupid) and we are not learning how these techniques actually work, but with your videos I will be ready to get a job.
much thanks for the clear illustration! One more question I have is that how the exchange occurs between the two elements of same charges ( B and C) in other way what is the reason behind the settlement of the protein of interest (C) that we want to eliminate and having the exchanged element (B) in our outflow??
Video is great thank you ! Tho, for the last part, the Salting out is not so clear. I didn't undersand why proteins with 1 nucleotide are eluted first.
Mam why we use Isopropyl alcohol or any organic modifier in Elution buffers during collection of Elute ( intereste protein) Actually I am using DEAE sepharoge fast flow resine for separate of lys-arg insulin from mixture. Here I am using 20mM Tris HCl+85mM Nacl+30%IPA? So why we use this IPA?
I would use anion exchanger( positively charged stationary phase), adjust my medium PH above the pka to deprotonate and make my Protein negative, I wouldn't want a lower pH that will make my Protein positive as there are higher chances of having other Proteins that are stable around that pH that can bind alongside my Protein. For elution, I will gradually decrease the inonic strength of my Buffer.
You need first to isolate bromate from drinking water using anion exchange chromatography, then you elute bromate using salting out. And then you can detect the presence of Bromate using a type of spectroscopy or UV light detector. There are machines nowadays that combine all these steps together, they are specially designed to detect bromate in drinking water.
7:50 totally wrong explanation. It is not true that in pI half of all molecules of aminoacid is in cationic form and half of all molecules is in anionic form.
you have no idea how you SAVED ME , much much love for you my savior
Thank you you explained that 2 substances are in ion column...subs A directly attached to resin and subs B that is bound to it by an ionic bond and that B is the one to be exchanged with the analyte...thank you for revealing this
For someone who has not enrolled in a Biochemistry laboratory class, this has helped me visualize what to do in the future. Thank you.
if the ph of the medium is lower than the pI of the protein, then the protein will be protonated and hence will be positively charged. If the ph is higher than pI then less proton are available in the medium and so the protein will lose its hydrogens from the side chains and become negatively charged.
Ya you are right
very good explanation! definitely helped me to prepare for my upcoming exams . thank you for this. keep this up, you're doing great job
Your videos are amazing..... please keep making such videos and try to make videos frequently....videos are very helpful
Another rule, is that you should use a pH at least 1 unit away from the PI in order to make sure the protein/peptide is completely protonated/deprotonated. So, if the PI is 2, and the pH is 2.3, you will probably not get good retention of your target peptide.
It makes your protein more soluble in the buffer
Thank you for the effort. Keep it up!
Wow!!! You are so awesome! This is the best video that explained this method so detailed and so understandable! This was so helpful! Thank you!
Thank you for these great videos! My biotech LAB classes are only virtual right now (which is stupid) and we are not learning how these techniques actually work, but with your videos I will be ready to get a job.
You can work for me where you from?
Great Video! Explained really well! Thank you so much
It's really helpful for me, add some other's techniques like HPLC, GC etc...
Thank you for the great and clear explanation
It was beautiful
I understood everything
Great job 👏
Thank you very much for explaining so good.
this really helped a lot! thank you !
Thank you for your clear explanation! 😊
Very nice presentation and highly useful...
thank you so much, it helped me a lot!
Great video. Thanks a lot🎉.
It's best video for this topic
Thanks a lot Mam very informative lecture
Super explaination
Wonderful!!!
Very informative video. How I can digest environmental samples to run it on IC ?
much thanks for the clear illustration! One more question I have is that how the exchange occurs between the two elements of same charges ( B and C) in other way what is the reason behind the settlement of the protein of interest (C) that we want to eliminate and having the exchanged element (B) in our outflow??
It can be because subs A has more affinity to C than B...or because the concentration of C is much higher than B
Excellent
You're the BEST
very well explained
THANK YOU SO MUCH
When do you use Ion or Absorption? Will the choice be subject to the substance of study?
please in ion chromotograph Which columns are the most preferred in this technique
How will B+ on anion exchange resin get replaced by our protein of interest?
Thankyou so much
It was really helpful, I have only one observation is that you talk aloud sometimes. But the explaination was clear and great for me. Thank you !
Thank you for the remark ... trier to solve the problem in my two new videos :)
very helpful
how to determine Ph of protein my protein has PI = 5.51 , what will be it's ph at 7.2 buffer .
you probably said reverse at 11:15
What defines what is Polar or non-polar?
Thanks
You have asked protein is -vly charged at ph less than 5 if pi of protein is 5
Video is great thank you ! Tho, for the last part, the Salting out is not so clear. I didn't undersand why proteins with 1 nucleotide are eluted first.
I’ll probably think it’s because it has less binding groups attached to the stationary phase coupled with its having the lowest molecular weight.
@ 11.15
if pH is less, than pI, it would be positive charge
When the Ph of the medium is less than the PI of the protein .. than the protein is definitely positively charged
what might be substance B?
Maam if ph of sol is less than 5 for pi5 then protein should be +vly charged here is confusion
yes. so where is the confusion?
Mam why we use Isopropyl alcohol or any organic modifier in Elution buffers during collection of Elute ( intereste protein)
Actually I am using DEAE sepharoge fast flow resine for separate of lys-arg insulin from mixture.
Here I am using 20mM Tris HCl+85mM Nacl+30%IPA?
So why we use this IPA?
is there any differences of chromatogram for cation and anion ion exchange chromatography?
No difference in the chromatogram for inion and cation exchange chromatography ..
@@biomedicalandbiologicalsci4989 Hi. Thanks for the wonderful video. But at place Anion has been written as Inion.
It’s really helpful, but isn’t it “anion” not “inion”?
what reaction takes place in this separations
ion exchange
Have you heard of "Single-displacement reaction?"
If pka is 5...then what method should be used for chromatography and what ph should be used for elution
I would use anion exchanger( positively charged stationary phase), adjust my medium PH above the pka to deprotonate and make my Protein negative, I wouldn't want a lower pH that will make my Protein positive as there are higher chances of having other Proteins that are stable around that pH that can bind alongside my Protein. For elution, I will gradually decrease the inonic strength of my Buffer.
i want to test bromate by my drinking water then how i do it?
You need first to isolate bromate from drinking water using anion exchange chromatography, then you elute bromate using salting out. And then you can detect the presence of Bromate using a type of spectroscopy or UV light detector. There are machines nowadays that combine all these steps together, they are specially designed to detect bromate in drinking water.
@taimour anjum why u want to do that test?
Inion exchange chromatography? Check your slides before sharing them....
It is great but please when you talk can you step wiggling your mouth.
7:50 totally wrong explanation. It is not true that in pI half of all molecules of aminoacid is in cationic form and half of all molecules is in anionic form.
So bad 😔
Wonderful!!!