Protein Purification - Pouring and Packing an Agarose Column
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- Опубликовано: 12 сен 2024
- GoldBio provides a selection of prepacked resins; however, packing your own columns can be a more cost-effective approach. We offer a variety of empty columns for your specific needs. This protein purification video will demonstrate the methods used to pack a bulk agarose affinity resin into a reusable purification column. After the column is poured it will be ready for immediate use or for storage. When storing your column, be sure to keep it upright at 4 °C. While it has a shelf life of approximately four weeks, we recommend that you only pack columns that will be used within one week
Necessary Supplies:
Bulk affinity resin
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De-ionized Water
EquiIibration/Bindìng/ or Wash buffer (with low imidazole concentration)
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Empty plastic column with bottom and top Stoppers www.goldbio.co...
A beaker or tube to catch flow through
A ring stand and clamp
Small funnel for head of column
Degas the agarose bead resin and buffer completely prior to adding anything into
the column.
Add one column volume of Resin Wash Buffer to the empty column and slowly drip buffer through the column to remove air bubbles from under frit. If air bubbles remain, tap the column to remove them. If air bubbles remain, the column can be centrifuged at a low RPM to
remove them.
Gently shake the bottle to obtain a homogenous suspension of Affinity Agarose
Beads (Ni, Co, Cu, Zn or Metal Free). Place a funnel in the head of column, then
open the bottom cap to start the flow of buffer through the column. Slowly run
the Agarose bead suspension down the walls of the column. Run the column
and continue to pour the suspension into the column until the desired column
bed height is reached. Do not allow the beads to completely settle in the
column.The best columns are made from a continuous pour. If you inadvertently
allow the beads to settle, use a pipette to mix the top of the settled matrix, then
continue to add new Agarose beads to the column. Do not allow the column to
run dry. If the column runs dry, you must repour the column. If you are using a
flow adapter, insert the adapter into the column head until it begins to displace
the liquid and be sure that no air is trapped under the sintered disc of the flow
adapter.
Wash the preservative off the resin by adding 5-10 column volumes of Buffer or
DI-HZO and running it through the column, again making sure the column doesn't
run dry.
If using the column immediately, move on to equilibrating the column. If storing
the column, add a little less than one column volume buffer or Di-HZO, place the
bottom and top caps, and store upright at 4°C. To prevent microbial growth, you
can add sodium azide to a final concentration of 0.02%
Equilibrating the Column
Equilibrate the column by adding 5 to 10 column volumes of Ni/Cu/Co Resin
Wash Buffer. Make sure to degas all the solutions before adding to the column
to avoid the formation of bubbles.
Running the column
Load the lysate (which contains the solubilized 6x histidine labeled protein) onto
the column. Control the flow rate of the addition of the lysate. We recommend
a binding flow rate of 12 mL per hour for a one mL column. Alternately you may
want to load the column with one third of the column volume and allow the
material to bind for five minutes prior to adding the next load of lysate.
Wash the column with 10 column volumes of Ni/Cu/Co Resin Wash Buffer. The
flow rate for a one mL column can be 30 mL per hour. Avoid compressing the
Agarose beads. Do not compress the Agarose beads by applying too much
pressure via a pump or a large pressure head.
Elute the protein using one of the following methods:
a. Apply a linear gradient of lOmM imidazole to SOOmlVl imidazole in Phosphate
Buffer, pH 8 (Gold Bio, Catalog #iP-500), or
b. Use a step gradient of Histìdine Protein Elution Buffer (elute with
SUOrnIVI, 400mm, and 500mm imidazole in Histìdine Protein Elution Buffer).
For step gradient, dilute Histìdine Protein Elution Buffer with Phosphate Buffer, pH 8:
100mM(50 mL) = 10 mL histidine : 40 mL phosphate
200mM (50 mL) = 20 mL histidine :30 mL phosphate
300mM (50 mL) = 30 mL histidine :20 mL phosphate
400mM (50 mL) = 40 mL histìdine : 10 mL phosphate
Regardless of which method you use for elution, you should collect fractions that are
commensurate with the column bed volume. The protein
should theoretically elute in the included volume of the column, which is
approximately equivalent to two thirds of the column bed volume.
Thanks for the video! Easy and clear "how to" protocol. It was a very big help when I was making my first self-packed column using chitin-beads. Hoping for more videos! Greetings from germany!!!
really helpful
Wow. This is really helpful. Just wandering whether it is possible for us to make the resin beans in lab scales?
How do you degas (small volumes)?
Can i desalt my protein with this technic..?
How to degas the resin?
What do you do with this I’m so confused?
We can purify proteins
What happens if the resin go dry? Thanks in advance.
Interesting. You asked this questions 1 year ago and no answer from them. Disappointing.
the resin would likely crack and break (you can see as they float on top of the liquid, rather than sink to the bottom). Not sure if that severely affect the function
During undergrad I packed my own protein purification columns and used those tiny columns to purify a protein from 50 ml of liquid. I was sleep deprived by the time I got to dialysis.
@@UNEARTHED36 really what you said 😅🤣
@@mouhamedosamaanbarji1896 It was like watching paint dry!
Where to get the resin
Can purchase from thermosfischer scientific!
@@deeptiyadav1403 its too expensive for me...
I am trying to to purify the alkaline (serine) protese from culture broth......if you have any idea how should I do it...plz share your views
@@swananddeshmukh9839 i am not an expert on protein purification..though u can use the "research gate" plateform or just see this paper "purification and partial characterization of thermostable serine alkaline protease from a newly isolatef bacillus subtilis PE-11"
@@deeptiyadav1403 Thank you