When the buffer is equilibrating the column, we count the number of drops it takes to get to 10 mL. Then we divide by 10 to get the number of drops for 1 mL. Students collect 1 mL aliquots into their tubes by counting the drops per tube.
Hello Dear 👋 Thank you for good tutorials actually I have one question is there any different between Gel Filtration LH-20 and HiTrap Desalting because both packed with G-25 and for purification secondary metabolite I need LH-20 but HiTrap more convinced I will appreciate if you give me good answers because I am working on unknown components
Hi, this is a very helpful video. I had a question. What is the best way to tell which fraction contains the protein if the everything we are dealing with is colorless?
Absorbance at 280 nm is commonly used to follow protein concentration. A protein assay (e.g. Bradford assay) can also be used to determine protein concentration.
Thank you so much, I really did understand. Please is there another video for calculations as to help one plot a graph? If there is one, I will be waiting. Thank you once again
Such a helpful demonstration. Could u help me with few queries.. Does the flow rate of buffer hampers the separation of protein? What should be the flow rate and what amount of protein can we load into the resin?
One of the best videos regarding size exclusion chromatography. I have some questions if you do not mind. I have bought Superdex 75 resin. If am not mistaken, I should pour the resin and let it for one or two days in a falcon tube eg to calculate the slurry (if I pour 10 mL of the resin and the new measurement is 5.5 mL it would be 55% of slurry). And then add this slurry into the column. My question is if I understood that correctly. Also, my column can host up to 16 mL, what is the preferred bed height to apply? I saw you have written about a 1:12 ratio of volumes. Finally, after packing a column, how many times can it be reused? Thank you in advance
Hi, I used sephadex G25 and packed the column similar to what's shown in the video. However, fluorescein doesn't retain as in the video but travels right though. Do you apply pressure in packing the column?
This was helpful, as there was no protocol on the GE website, you mention that 800 uL was a bit too much for this column, what volume of column per volume of sample is the limit?
About 20 mL of gel bed for each 1 mL of loaded sample is a good ratio to use. In this video, the gel bed is about 10 mL for 0.80 mL of loaded sample (12 to 1 ratio)
Can anybody recommend a process to purify an Oligonucleotide Mix in RNase Free Water. These oligos were only standard desalted. The volumes vary from 250mL to 1400mL. thank you Spencer
I am using Sephadex LH20 to separate and purify proanthocyanidins from wine. Methanol is the solvent. Bed volume 290 ml, with 4ml/g of dry material used for given solvent. How big should my sample be without causing poor seperation/resolution? Thanks!
Hello. My lab manual says to "Pour a column (Sephadex G-75 slurry) so that the bed is about 13 cm in height. Equilibrate the column in the elution buffer." Would equilibrating the column be what you did from 1:45 to 2:13?
Equilibration means allowing buffer to flow through the column so that the entire gel bed is at a constant pH and ion concentration. Adding two or three column volumes of buffer to the top of the gel bed and letting it run through will ensure that the buffer concentration is uniform throughout.
My gel (Sephadex G-100) keep running through out the columm. I dont know if it is the consistency or maybe the buffer I used to swell it up (sodium acetate 50mM pH 5,0 - the enzime Im looking to purify is well adapted to the this buffer) is not appropriate for it. Should I plug a small piece of cotton to the botton of my columm and then add the sephadex to avoid the gel to leak out?
If the gel leaks out of the column, you will need to add something like cotton or glass wool to keep the beads in. The columns I use have built-in frits that retain the fine gel beads.
When refrigerated, the gel can last at least a week, maybe a few weeks. If refrigerated with an azide-containing buffer, the gel can last for a year or so (0.1% sodium azide).
The most useful video on filtration columns!
Awesome awesome .
I was reading the textbook and not getting it .
But after watching it whole stuff is clear .
Thank you much ❤️ for this video.
I absolutely love this video! Thank you!
Very clear demonstration. Thank you
Thanks! This is incredibly helpful
Excellent video! Thank you!
This is a very useful explanation. Thanks.
If anything catches on fire at the lab tonight, i will blame you cause i will follow your method.
Very nice demonstration 👏❤️..Thank you🙏
Thank you, you made my day 😊😊
Very nice video presentation!
Very nice demonstration..Thank you.
How do you know when to swap the test tube drain caches?
When the buffer is equilibrating the column, we count the number of drops it takes to get to 10 mL. Then we divide by 10 to get the number of drops for 1 mL. Students collect 1 mL aliquots into their tubes by counting the drops per tube.
very nicely explained thank u so much!!!
Hello Dear 👋
Thank you for good tutorials actually I have one question is there any different between Gel Filtration LH-20 and HiTrap Desalting because both packed with G-25 and for purification secondary metabolite I need LH-20 but HiTrap more convinced I will appreciate if you give me good answers because I am working on unknown components
amazing
Hi, this is a very helpful video. I had a question. What is the best way to tell which fraction contains the protein if the everything we are dealing with is colorless?
Absorbance at 280 nm is commonly used to follow protein concentration. A protein assay (e.g. Bradford assay) can also be used to determine protein concentration.
Uv spectrometry
Thank you so much for this 😄😄😄
thanks for tutorial!
I take plant extract, i want different component of plant extract, which method is useful and accurate to separate plant extract component?
Thank you so much, I really did understand. Please is there another video for calculations as to help one plot a graph? If there is one, I will be waiting. Thank you once again
s it possible to separate the sizes of DNA?
thanks for your help
Nicely explained
Thank you!!!
This vid is very help, thank you so much!
Such a helpful demonstration.
Could u help me with few queries..
Does the flow rate of buffer hampers the separation of protein? What should be the flow rate and what amount of protein can we load into the resin?
would the steps be the same (+ the buffer used) for Sephadex LH20?
what solvent did you put the 9 grams of sepahdex in? Was it water or alcohol?
thank you
Thank you!
Neeru ma'am bought me here.
Good day, sir. Can I ask what kind pf column is that? Can I use the conventional glass open column (similar to those used for silica gel)?
What's name of buffer
May l know the concentration of both the dyes .
I was also interested in the concentration per dye.
Lovely
Can you please guide me how can I purify a protein of MW 20-25 KDa using chromatography from a conjugation buffer
Nice
One of the best videos regarding size exclusion chromatography. I have some questions if you do not mind.
I have bought Superdex 75 resin. If am not mistaken, I should pour the resin and let it for one or two days in a falcon tube eg to calculate the slurry (if I pour 10 mL of the resin and the new measurement is 5.5 mL it would be 55% of slurry). And then add this slurry into the column. My question is if I understood that correctly.
Also, my column can host up to 16 mL, what is the preferred bed height to apply? I saw you have written about a 1:12 ratio of volumes.
Finally, after packing a column, how many times can it be reused?
Thank you in advance
But its a pre-made column right?
What is the column you are using? Thank you.
Hi, I used sephadex G25 and packed the column similar to what's shown in the video. However, fluorescein doesn't retain as in the video but travels right though. Do you apply pressure in packing the column?
Thank you so much for making this video
Why does the larger molecule move faster?
How to make blue dextran.. We should dissolve into distilled water or in phosphate buffer plz tell me
Its very useful.
Thanks for your nice video. What is the name of the empty column you used?
Thank you sooo muchhh❤️❤️❤️
Thankuuuu
Can I use distilled water in replace of tris buffer?
Thank you, :)
How to extract ricin protein from this process??
What kind of column is that?
what if you drink it?
This was helpful, as there was no protocol on the GE website, you mention that 800 uL was a bit too much for this column, what volume of column per volume of sample is the limit?
About 20 mL of gel bed for each 1 mL of loaded sample is a good ratio to use. In this video, the gel bed is about 10 mL for 0.80 mL of loaded sample (12 to 1 ratio)
Can anyone help me to know: What's the scientific name of this type of tubes used for this purpose? Thanks
Can anybody recommend a process to purify an Oligonucleotide Mix in RNase Free Water. These oligos were only standard desalted. The volumes vary from 250mL to 1400mL.
thank you
Spencer
Can you please guide me how I can purify a protein of mw 40kd using chromatography from a fermented broth.
Thank you this is a huge help! How much buffer did you swell 9g of sephadex in, in the beaker?
About 20 mL of buffer is recommended for each gram of resin.
Biochem squad
I am using Sephadex LH20 to separate and purify proanthocyanidins from wine.
Methanol is the solvent. Bed volume 290 ml, with 4ml/g of dry material used for given solvent. How big should my sample be without causing poor seperation/resolution?
Thanks!
The recommended sample volume would be 1-2% of the total bed volume according to the manufacturer.
Thank u sir
Hello. My lab manual says to "Pour a column (Sephadex G-75 slurry) so that the bed is about 13 cm in height. Equilibrate the column in the elution buffer." Would equilibrating the column be what you did from 1:45 to 2:13?
Equilibration means allowing buffer to flow through the column so that the entire gel bed is at a constant pH and ion concentration. Adding two or three column volumes of buffer to the top of the gel bed and letting it run through will ensure that the buffer concentration is uniform throughout.
@@acr92651 I take plant extract, i want different component of plant extract, which method is useful and accurate to separate plant extract component?
nice, it will help :)
My gel (Sephadex G-100) keep running through out the columm. I dont know if it is the consistency or maybe the buffer I used to swell it up (sodium acetate 50mM pH 5,0 - the enzime Im looking to purify is well adapted to the this buffer) is not appropriate for it. Should I plug a small piece of cotton to the botton of my columm and then add the sephadex to avoid the gel to leak out?
If the gel leaks out of the column, you will need to add something like cotton or glass wool to keep the beads in. The columns I use have built-in frits that retain the fine gel beads.
@@acr92651 Can you please let me know the scientific name of the column tubes you have used. I would like to purchase the same.. Thanks
What's the expiry date of the packed gel in the column?
When refrigerated, the gel can last at least a week, maybe a few weeks. If refrigerated with an azide-containing buffer, the gel can last for a year or so (0.1% sodium azide).
useful
dank goethe uni so viele views
sigma sephadex
what was the buffer you used?
30 mM Tris, pH 8.0
To many bubbles!
But still a nice video ✌
wer ist hier wegen dem Biochemie Praktikum hahah?