How to do FPLC - an example of affinity chromatography
29.6 هزار بار بازدید -
3 سال پیش
-
Fast Protein Liquid Chromatography, short
Fast Protein Liquid Chromatography, short FPLC, is a commonly used for purification of biomolecules, including proteins via affinity chromatography.
Due to the setup of hardware and the involvement of settings via a computer software, the method can be intimidating on the first try.
However FPLC provides the opportunity for maximum control of all steps of your chromatography, even miniscule modifications are possible.
Therefore, we created this video to explain a FPLC: We used the example of affinity chromatography by purifying His-tagged GFP using our in-house developed Ni-INDIGO Agarose resin.
Ni-INDIGO resin can be found here:
https://cube-biotech.com/products/pro...
And pre-packed into FPLC Ready Columns/Cartridges:
https://cube-biotech.com/products/pro...
The recipes for all mentioned buffers can be found here:
https://cube-biotech.com/products/pro...
Time stamps of the shown procedure:
0:00 - Intro
0:11 - Build-up of a FPLC system
1:15 - How to set-up a FPLC - Buffers & Columns
1:58 - How to configure the FPLC Software (Biologic Duoflow Example)
3:22 - The chromatogram - Explained!
4:27 - Fractioning & Collecting sample
4:50 - Endcard
The individual steps in order:
1. Attach binding and elution buffer to your system
2. Unplug top of the affinity resin cartridge and plug it in.
IMPORTANT: Ensure that not air gets into the system.
3. Do the same with the bottom plug of the affinity resin cartridge
4. Load your protein sample into the loop
5. Setup of the program - The amounts set in the protocol are optimized for His-tag GFP purification. But it can be used as a starting point for the setting of other His-tag protein purifications with His-tag affinity cartridges:
The program consists of three different steps varying in their ratio between binding and elution Buffer.
5.1 Step A - Binding: 100% binding buffer and 0% elution buffer
5.2 Step B - Washing: 92% binding buffer and 8% elution buffer
5.3 Step C - Elution: 0% binding buffer and 100% elution buffer
5.4 Afterwards set up the volumes of the elution fractions.
6. Collect the fractions which contain your purified protein.
Due to the setup of hardware and the involvement of settings via a computer software, the method can be intimidating on the first try.
However FPLC provides the opportunity for maximum control of all steps of your chromatography, even miniscule modifications are possible.
Therefore, we created this video to explain a FPLC: We used the example of affinity chromatography by purifying His-tagged GFP using our in-house developed Ni-INDIGO Agarose resin.
Ni-INDIGO resin can be found here:
https://cube-biotech.com/products/pro...
And pre-packed into FPLC Ready Columns/Cartridges:
https://cube-biotech.com/products/pro...
The recipes for all mentioned buffers can be found here:
https://cube-biotech.com/products/pro...
Time stamps of the shown procedure:
0:00 - Intro
0:11 - Build-up of a FPLC system
1:15 - How to set-up a FPLC - Buffers & Columns
1:58 - How to configure the FPLC Software (Biologic Duoflow Example)
3:22 - The chromatogram - Explained!
4:27 - Fractioning & Collecting sample
4:50 - Endcard
The individual steps in order:
1. Attach binding and elution buffer to your system
2. Unplug top of the affinity resin cartridge and plug it in.
IMPORTANT: Ensure that not air gets into the system.
3. Do the same with the bottom plug of the affinity resin cartridge
4. Load your protein sample into the loop
5. Setup of the program - The amounts set in the protocol are optimized for His-tag GFP purification. But it can be used as a starting point for the setting of other His-tag protein purifications with His-tag affinity cartridges:
The program consists of three different steps varying in their ratio between binding and elution Buffer.
5.1 Step A - Binding: 100% binding buffer and 0% elution buffer
5.2 Step B - Washing: 92% binding buffer and 8% elution buffer
5.3 Step C - Elution: 0% binding buffer and 100% elution buffer
5.4 Afterwards set up the volumes of the elution fractions.
6. Collect the fractions which contain your purified protein.
3 سال پیش
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