Thank you so much for such an informative video! I have a PhD in Chemistry (but in solid state chemistry) and have recently switched to a new role where i will need to do some peptide synthesis and this video is absolutely amazing for me!
Hi Steve, great video, just wondering after incubating the resin with 95% TFA for 2 hours, how did you remove beads? did you filter it? if yes, what type of filter did you used?
Great video it mean a lot for me 'cause right mow I am working on the synthesis of peptide so could you provide some paper regarding your peptide synthesis it can help me a lot
Great video! You gave me a good idea of how SPPS works. I have a question: Do you reuse the resin for another peptide synthesis? Or you have to use new resin for every batch?
Hii there, nice video. I am bit confused on my synthesis as I got a sequence using phage display library against a pathogen for example: STFSQNG (N to C) and I want to synthesise it using Fmoc SPPS. But as the FMOC SPPS using Rink amide resins generate from C to N, should I use Glycine as first amino acid (reverse) or use S (serine) as first amino acid and then T,F,S etc.
good video, there is very little useful information out there when running peptide synthesis. even device manufacturers do not give in detail information.
Hey there! It's been quite awhile since you posted this video -- hopefully you get to see this! I'm currently doing SPPS as an undergraduate project. The research assistant in this lab group washes with DMF after deprotection with piperidine but there is no significant activation process for the amino acid prior to its addition to the reaction vessel for attachment to the resin. They simply add HATU and then immediately following DIEA the amino acid HATU DIEA solution is poured into the column. It was made out to be a big deal that I have to be very quick from the point of DIEA addition to pouring the solution into the vessel to soak the resin. I am wondering if this is inaccurate, as you pre-activate your amino acids for 5 minutes, I can't imagine it is important to be so fast. I've spent a while trying to find information in the literature but I can't find anything about amino acid activation time with DIEA just that it's used in the coupling process and no further information. I'd really like to hear back if you have the time. Thanks!
My lab also washes with DMF (and DCM) after deprotection. I was actually told the opposite from what you are told. The instructions told me to specifically wait at least a minute (if not more) after adding DIEA to the amino acid and HATU concoction to let it activate before adding the DIEA + amino acid + HATU mixture to the reaction vessel. I don't know if this helped
All my work is devoted to differential algebra: searching for exact solutions to PDEs and ODEs with a particular interest in application to biochemistry. I want to know if there is some way we can massively scale up the Bruce Merrifield solid phase peptide synthesis process to produce edible proteins: i.e. meat.
You can check out any standard literature. The convention is to use 3 eq Fmoc-AA-OH with 6 eq DIPEA in minimum amount of DMF, 3 eq HATU (along with HOAt if you want)in minimum amount of DMF. (DMF is almost always replacable completely with NMP but it is not advised to change solvents during synthesis).
After incubating the resin with 95% TFA for 2 hours, I eluted the TFA solution and used a rotovap to remove the solvent. Then I dissolved the peptide in water and lyophilized to achieve a solid product. Some peptides can be precipitated from the TFA solution by using ether.
I am Steve Sogo of Laguna Beach High School. I received my graduate school training at Caltech. I run a Chemical Research class with seniors (12th grade students).
@@acr92651 Thank you! Holy moly, they teach this stuff in high school now? My chemistry laboratory experience in Cinnaminson High School, NJ 1980 to 1982 was very good, but never THIS good!
Awesome video, thanks for share. I have a question. Have you ever perform TFA cleavage with Wang or Merrifield resin? is the same process than aminoacids? peace.
TFA works with Wang resin, but Merrifield resin uses a different method. This website may help you choose the proper cleaving agent: www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Aldrich/Brochure/al_chemfile_v3_no4.pdf
The red peptide shown in this video, bearing the Dabcyl group, absorbs light at a long wavelength. Many peptides that do not have special groups must be detected at 214 nm, a wavelength at which peptide bonds (amides) have significant absorbance.
For HPLC, I use water + acetonitrile, each containing 0.1% trifluoroacetic acid. This is a common system for peptide analysis. I purchase my solvents (with TFA already included) from Fisher Scientific or Sigma-Aldrich.
A question, when you order custom-made peptides from companies like ThermoFisher or Sigma, is this process performed manually like in the video or automated?
Thank you so much for such an informative video! I have a PhD in Chemistry (but in solid state chemistry) and have recently switched to a new role where i will need to do some peptide synthesis and this video is absolutely amazing for me!
Hey Steve! Amazing video! Suoer clear and informative. This is exactly what I was looking for! I'm starting a new role in peptide synthesis 😊
Hi Steve, great video, just wondering after incubating the resin with 95% TFA for 2 hours, how did you remove beads? did you filter it? if yes, what type of filter did you used?
Great video it mean a lot for me 'cause right mow I am working on the synthesis of peptide so could you provide some paper regarding your peptide synthesis it can help me a lot
Great video! You gave me a good idea of how SPPS works.
I have a question: Do you reuse the resin for another peptide synthesis? Or you have to use new resin for every batch?
Nope the resin is done after one use.
Hi , thanks you for this video, can you please explain why you have red color when you add the first 20% piperidin to Rink amineResin ??
Hi! Thank you for this great video. I have a question. How do you elute the TFA solution, please? Thank you.
Hi Steve, will we use Kaiser test for deprotection
Also in carbetocine the colour of Peptide is red during TFA cleavage deprotection
Hii there, nice video. I am bit confused on my synthesis as I got a sequence using phage display library against a pathogen for example: STFSQNG (N to C) and I want to synthesise it using Fmoc SPPS. But as the FMOC SPPS using Rink amide resins generate from C to N, should I use Glycine as first amino acid (reverse) or use S (serine) as first amino acid and then T,F,S etc.
Could you tell me if these polypropylene vessels are reusable?
good video, there is very little useful information out there when running peptide synthesis. even device manufacturers do not give in detail information.
Can you guide some about alanine and hydroxy proline ..dipeptide
Hey there! It's been quite awhile since you posted this video -- hopefully you get to see this! I'm currently doing SPPS as an undergraduate project. The research assistant in this lab group washes with DMF after deprotection with piperidine but there is no significant activation process for the amino acid prior to its addition to the reaction vessel for attachment to the resin. They simply add HATU and then immediately following DIEA the amino acid HATU DIEA solution is poured into the column.
It was made out to be a big deal that I have to be very quick from the point of DIEA addition to pouring the solution into the vessel to soak the resin. I am wondering if this is inaccurate, as you pre-activate your amino acids for 5 minutes, I can't imagine it is important to be so fast. I've spent a while trying to find information in the literature but I can't find anything about amino acid activation time with DIEA just that it's used in the coupling process and no further information. I'd really like to hear back if you have the time. Thanks!
My lab also washes with DMF (and DCM) after deprotection. I was actually told the opposite from what you are told. The instructions told me to specifically wait at least a minute (if not more) after adding DIEA to the amino acid and HATU concoction to let it activate before adding the DIEA + amino acid + HATU mixture to the reaction vessel. I don't know if this helped
All my work is devoted to differential algebra: searching for exact solutions to PDEs and ODEs with a particular interest in application to biochemistry. I want to know if there is some way we can massively scale up the Bruce Merrifield solid phase peptide synthesis process
to produce edible proteins: i.e. meat.
It wouldn't currently be viable to produce meat this way. There is a huge environmental impact in SPPS due to the solvents used i.e. DMF.
Hi! Thanks for this great video! I have a question. How do you dissolve amino acids that have low solubility in DMF for example phenylalanine?
use NMP, it also dissolves Histidine, tyrosine and proline
sorry!what is the valence of rink amide resin? for changing meq to mg
Can you link the literature used to determine the equivalents of amino acid, HATU, DIEA, and NMP used in the coupling solution?
You can check out any standard literature. The convention is to use 3 eq Fmoc-AA-OH with 6 eq DIPEA in minimum amount of DMF, 3 eq HATU (along with HOAt if you want)in minimum amount of DMF. (DMF is almost always replacable completely with NMP but it is not advised to change solvents during synthesis).
Hi! Thanks for this great video! I have a question. What have you done after putting your resin into TFA solution?
After incubating the resin with 95% TFA for 2 hours, I eluted the TFA solution and used a rotovap to remove the solvent. Then I dissolved the peptide in water and lyophilized to achieve a solid product. Some peptides can be precipitated from the TFA solution by using ether.
Who are you? What is your name: the person physically doing what we are seeing? Who is talking?
What institution? Where? Thanks.
I am Steve Sogo of Laguna Beach High School. I received my graduate school training at Caltech. I run a Chemical Research class with seniors (12th grade students).
@@acr92651 Thank you! Holy moly, they teach this stuff in high school now? My chemistry laboratory experience in Cinnaminson High School, NJ 1980 to 1982 was very good, but never THIS good!
Awesome video, thanks for share. I have a question. Have you ever perform TFA cleavage with Wang or Merrifield resin? is the same process than aminoacids? peace.
TFA works with Wang resin, but Merrifield resin uses a different method. This website may help you choose the proper cleaving agent:
www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Aldrich/Brochure/al_chemfile_v3_no4.pdf
thank you so much for your help!
cool
I need this lab FOXO-4 natural
Why don't u commercialize yr video by introducing ads on youtube
Thanks for this Awesome video! I have a question. Did you use 510 nm wavelength to detect in HPLC analysis, and why? Do you suggest 214 nm?
The red peptide shown in this video, bearing the Dabcyl group, absorbs light at a long wavelength. Many peptides that do not have special groups must be detected at 214 nm, a wavelength at which peptide bonds (amides) have significant absorbance.
May i ask what kind of mobile pahse you used at the HPLC?
I am going to recreate some of the experiments i have seen you do.
For HPLC, I use water + acetonitrile, each containing 0.1% trifluoroacetic acid. This is a common system for peptide analysis. I purchase my solvents (with TFA already included) from Fisher Scientific or Sigma-Aldrich.
A question, when you order custom-made peptides from companies like ThermoFisher or Sigma, is this process performed manually like in the video or automated?
Usually automated. And usually it is done using microwave synthesis which is much quicker.