I am trying to purify tagged protein by affinity purification. The problem is I need large quantity of protein but with the kit purified volume is 30 ul only. Any suggestions how to get larger quantity and volume of purified protein?
TIA.
Kakali Das
You have to scale up by using more of everything. Often, kits come in different sizes for different scales of work.
You mean you have 30ul of affinity resin? The volume is not so important, capacity is what you need to consider, though it seems likely that "a large quantity of protein" is more than 30ul of resin will bind.
Let's say you eed 'y' mg. Find out the binding capacity of the affinity resin (mg per packed ml -the supplier should be able to tell you this). Calculate the amount of packed resin you need to bind 'y' mg of protein. Personally, I'd use 25% more resin just to give some safety margin. If 30ul of resin is too little, as seems likely, then you need to scale up as already mentioned by Adam B Shapiro .
The other thing you need to think about is how much tagged protein you actually have in your crude extract. Even if you want a large amount of protein, there is no point in making an affinity column whose capacity vastly exceeds the amount of tagged protein in the sample. Remember too, you will not get 100% recovery, hence if you want to recover 'y' mg you might need as much as 1.5y mg in the crude extract.
Kakali Das As mentioned above, you have to scale up the process to increase yield. If kit has limitation, you have to manually develop the affinity purification, so with good volume of initial culture and resin, you can get higher yield. Thank you.
As the others stated you have to Scale up.
If you still want to use Kits I would recommend the HisCube MIDI Kit:
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/indigo-ni/770/hiscube-ni-indigo-his-tag-protein-purification-midi-kit
I just assumed that you use His-tag based purification as it is the one with the most yield.
Dear Kakali Das
which kind of affinity purification ? IMAC? proteinA? ProtG? GST=? MBP?
i tihnk that the best way is buy bulk resin, empty coloumns suitable for gravity flow, prepare your buffer and use the culture and resin scale that fit better with the the amount of protein that you would like to purify
for example in the following link
https://proteocool.blogspot.com/2021/05/tips-of-week7small-scale-imac.html
you found some examples of different gravity flow purification scale thi'm using for purification of his tagged protein by IMAC.
ciao
Manuele
Additional information is required to provide any meaningful guidance.
Expression system? Info on protein would be helpful. Which tag are you using? Which purification system are you using? Do you have any biochemists in your dept, if so they would be a good place to start. Otherwise we are happy to guide you.
Thank you all for valuable suggestions. Finally I could purify protein by affinity chromatography
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