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.
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.
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
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.
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.
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?
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.
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
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
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
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?
For someone who has not enrolled in a Biochemistry laboratory class, this has helped me visualize what to do in the future. Thank you.
you have no idea how you SAVED ME , much much love for you my savior
Very nice presentation and highly useful...
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!
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
Thank you for your clear explanation! 😊
It's really helpful for me, add some other's techniques like HPLC, GC etc...
It was beautiful
I understood everything
Great job 👏
Your videos are amazing..... please keep making such videos and try to make videos frequently....videos are very helpful
very good explanation! definitely helped me to prepare for my upcoming exams . thank you for this. keep this up, you're doing great job
Great video. Thanks a lot🎉.
@ 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
Thank you for the effort. Keep it up!
Thank you very much for explaining so good.
Super explaination
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 :)
Great Video! Explained really well! Thank you so much
It's best video for this topic
you probably said reverse at 11:15
Thank you for the great and clear explanation
Excellent
Thanks a lot Mam very informative lecture
When do you use Ion or Absorption? Will the choice be subject to the substance of study?
You're the BEST
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.
this really helped a lot! thank you !
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?
very well explained
You have asked protein is -vly charged at ph less than 5 if pi of protein is 5
Very informative video. How I can digest environmental samples to run it on IC ?
thank you so much, it helped me a lot!
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?
how to determine Ph of protein my protein has PI = 5.51 , what will be it's ph at 7.2 buffer .
What defines what is Polar or non-polar?
It’s really helpful, but isn’t it “anion” not “inion”?
very helpful
Wonderful!!!
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.
Thankyou so much
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.
THANK YOU SO MUCH
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?
what might be substance B?
Thanks
what reaction takes place in this separations
ion exchange
Have you heard of "Single-displacement reaction?"
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!!!