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Introduction to desalting and buffer
exchange of protein samples
Why desalting and buffer exchange?
• Where are desalting/buffer exchange used in the protein purification workflow?
• What...
Why desalting and buffer exchange?
Desalting/buffer exchange can be used:
• Before protein purification
• Between protein purification steps
• After protein ...
Desalting
• It is the removal of salts
• In desalting, the column
is equilibrated with
water or buffer without
salt
Desalt...
Goal of desalting/buffer exchange:
make the sample compatible with the next step
• Purification steps to remove a low-mole...
As desalting/buffer exchange is size
exclusion chromatography (SEC), you do not
need to add a separate desalting/buffer
ex...
Desalting/buffer exchange has many advantages over dialysis
including reduced time and high recovery
• Fast - helps preser...
Desalting and buffer exchange.
Typical applications
SDS does not tolerate high levels of salt
High ionic strength has been shown to
reduce the amount of SDS bound to
proteins...
Imidazole considerably reduces labeling
efficiency
It is important to clean-up protein
samples containing imidazole (histi...
Resins and columns for desalting and
buffer exchange
Did you know?
One late night during 1957, as a young scientist
was leaving the lab, he made a mistake.
The mistake would b...
Two types of SephadexTM resins
Suitable for rapid desalting and
buffer exchange of biomolecules
with a molecular weight > ...
Overview of columns from GE
for desalting/buffer exchange
Benefit of centrifugation:
The sample will not be diluted:
• No ...
• The pack includes four adapters for centrifugation
PD-10 Desalting columns: ready-to-use for convenient gravity-flow
or ...
HiTrap™ Desalting columns: easy-to-use with syringe, peristaltic
pump, or chromatography system
KA1392071117PP 17
Visit pr...
Tip! Connect your columns in series to handle larger sample sizes
with chromatography systems
KA1392071117PP
To minimize d...
Tired from manual desalting of your protein sample?
ÄKTA™ start system makes an easy and economical transition to automate...
Learn more
Want to learn more or discover GE’s desalting columns?
KA1392071117PP
This handbook contains
principles and methods
about ...
Thank you
Visit us at
protein.gelifesciences.com
Or join ÄKTA™ club today
gelifesciences.com/AKTAclub
KA1392071117PP 22
GE, the GE Monogram, ÄKTA, HiPrep, HiTrap, HisTrap, MultiTrap, MidiTrap, MiniTrap, Sephadex, and SpinTrap are trademarks o...
Introduction to desalting and buffer exchange of protein samples
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Introduction to desalting and buffer exchange of protein samples

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This presentation is an introduction to desalting and buffer exchange of protein samples: What is it? What are the typical applications?
It also gives an overview of columns from GE for desalting and buffer exchange.

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Introduction to desalting and buffer exchange of protein samples

  1. 1. Introduction to desalting and buffer exchange of protein samples
  2. 2. Why desalting and buffer exchange? • Where are desalting/buffer exchange used in the protein purification workflow? • What is desalting/buffer exchange? • What is the goal of desalting/buffer exchange? • Comparing desalting/buffer exchange with dialysis Typical applications • Desalting before analysis by SDS-PAGE • Imidazole removal prior to labeling Resins and columns for desalting and buffer exchange • Sephadex™ resins • Columns for desalting/buffer exchange from GE Healthcare’s Life Sciences Business • Tip to handle large sample sizes • Automate your desalting step Learn more Content – click on the section of your choice KA1392071117PP 2
  3. 3. Why desalting and buffer exchange?
  4. 4. Desalting/buffer exchange can be used: • Before protein purification • Between protein purification steps • After protein purification • Before protein analysis Desalting and buffer exchange can be used in many steps of the protein purification workflow Visit proteins.gelifesciences.com >> KA1392071117PP Prepare Purify Analyse 4
  5. 5. Desalting • It is the removal of salts • In desalting, the column is equilibrated with water or buffer without salt Desalting and buffer exchange: what is it? • It is the replacement of one buffer with another • In buffer exchange, the column is equilibrated with the desired buffer Buffer exchange Desalting/buffer exchange are performed with columns that contain a size exclusion chromatography resin KA1392071117PP 5 Pooled fractions Removal of NaCl from albumin solution with PD-10 Desalting column. The albumin is eluted in volume fractions between 2.5 to 6.0 mL. Example
  6. 6. Goal of desalting/buffer exchange: make the sample compatible with the next step • Purification steps to remove a low-molecular weight component used for elution • Ion exchange chromatography to remove salt for the purpose of decreasing the ionic strenth of a sample to allow binding to the column • SDS-PAGE analysis • Removal of inhibitors from enzymes before studying them • Low-pH elution in a purification step, to restore pH • Protein purification to make a final adjustment of conditions of the purified protein • Ion exchange chromatography to remove salt used for elution • Labeling or other chemical modifications of a protein, to stop the reaction or to remove excess reagents Used before… Used after… Visit proteins.gelifesciences.com >> KA1392071117PP 6
  7. 7. As desalting/buffer exchange is size exclusion chromatography (SEC), you do not need to add a separate desalting/buffer exchange step directly after an SEC step in your workflow. Good to know! No need for desalting step directly after SEC step KA1392071117PP 7 Visit proteins.gelifesciences.com >>
  8. 8. Desalting/buffer exchange has many advantages over dialysis including reduced time and high recovery • Fast - helps preserve biological activity of the target protein • Does not require large volumes of buffer- less dilution • High recovery • Slow - usually done in the cold overnight • Dependent on diffusion of molecules- slower in the cold • Several replacements of the buffer needed for complete exchange • Not suitable for large sample volumes • Protein loss due to many steps Desalting/buffer exchange < 10 min Dialysis > 8 h KA1392071117PP Visit proteins.gelifesciences.com >> 8
  9. 9. Desalting and buffer exchange. Typical applications
  10. 10. SDS does not tolerate high levels of salt High ionic strength has been shown to reduce the amount of SDS bound to proteins, suggesting that high salt concentrations in the sample might interfere with molecular weight determination Desalting columns can be used to reduce salt concentration and improve SDS- PAGE results Importance of desalting before analysis by SDS-PAGE Visit proteins.gelifesciences.com >> KA1392071117PP 10
  11. 11. Imidazole considerably reduces labeling efficiency It is important to clean-up protein samples containing imidazole (histidine [his]-tagged protein) before labeling Buffer exchange step is used to improve labeling efficiency Importance of imidazole removal prior to labeling Visit proteins.gelifesciences.com >> KA1392071117PP 11
  12. 12. Resins and columns for desalting and buffer exchange
  13. 13. Did you know? One late night during 1957, as a young scientist was leaving the lab, he made a mistake. The mistake would be a turning point for his professor, Jerker Porath, and lead to the remarkable discovery of dextran-based Sephadex (SEparation PHArmacia DEXtran). Desalting and buffer exchange columns: it’s all about our SephadexTM size exclusion resin Visit proteins.gelifesciences.com >> KA1392071117PP 13
  14. 14. Two types of SephadexTM resins Suitable for rapid desalting and buffer exchange of biomolecules with a molecular weight > 5000 Recommended for rapid desalting and buffer exchange of samples like small proteins, peptides, and carbohydrates Separates biomolecules with a molecular weight > 700 from low molecular weight contaminants Sephadex G-25 Sephadex G-10 Visit proteins.gelifesciences.com >> KA1392071117PP 14
  15. 15. Overview of columns from GE for desalting/buffer exchange Benefit of centrifugation: The sample will not be diluted: • No concentration of the desalted sample is needed • Saves time and no loss of protein Format name SpinTrap™ MultiTrap™ MiniTrap™ MidiTrap™ PD-10 Desalting HiTrap™ HiPrep™ Sample volume ≤ 130 µL ≤ 130 µL ≤ 0.5 mL ≤ 1.0 mL ≤ 2.5 mL ≤ 1.5 mL ≤ 15 mL Application Screening and quick desalting of small sample quantities using a benchtop centrifuge High-throughput screening and small-scale purification using centrifuge or vacuum equipment Simple one-step desalting of proteins without centrifuge/vacuum equipment Simple one-step desalting of proteins without the need for equipment Simple one-step desalting of proteins without the need for additional equipment Easy-to-use with a syringe, peristaltic pump, or chromatography system Convenient scale- up. Preparative size exclusion chromatography CENTRIFUGATION CENTRIFUGATION (WITH BLUE ADAPTER) OR GRAVITY SYRINGE, PUMP, OR SYSTEM SYSTEM Visit proteins.gelifesciences.com >> KA1392071117PP 15
  16. 16. • The pack includes four adapters for centrifugation PD-10 Desalting columns: ready-to-use for convenient gravity-flow or centrifugation For gravity use For centrifugation Centrifugation adapters included • The box can be converted into a column stand • A buffer tray is included for easy disposal of flow- through buffer used during column equilibration Column stand Buffer tray KA1392071117PP 16 Visit proteins.gelifesciences.com >>
  17. 17. HiTrap™ Desalting columns: easy-to-use with syringe, peristaltic pump, or chromatography system KA1392071117PP 17 Visit proteins.gelifesciences.com >>
  18. 18. Tip! Connect your columns in series to handle larger sample sizes with chromatography systems KA1392071117PP To minimize dilution and still retain good separation, we recommend a sample volume that does not exceed 30% of the total resin bed volume. HiTrap™ Desalting HiPrep™ Desalting Maximu m sample volumes ≤ 1.5 mL per column ≤ 15 mL per column ≤ 7.5 mL if connected in series Up to five columns may be connected in series ≤ 60 mL if connected in series Up to four columns may be connected in series 18 Visit proteins.gelifesciences.com >>
  19. 19. Tired from manual desalting of your protein sample? ÄKTA™ start system makes an easy and economical transition to automated desalting KA1392071117PP Watch ÄKTA start video>> Download desalting protocol>> Discover ÄKTA start in details >>Compatible with HiPrep™ Desalting and HiTrap™ Desalting columns 19
  20. 20. Learn more
  21. 21. Want to learn more or discover GE’s desalting columns? KA1392071117PP This handbook contains principles and methods about size exclusion chromatography, and desalting Download handbook >> Visit proteins.gelifesciences.com >> 21
  22. 22. Thank you Visit us at protein.gelifesciences.com Or join ÄKTA™ club today gelifesciences.com/AKTAclub KA1392071117PP 22
  23. 23. GE, the GE Monogram, ÄKTA, HiPrep, HiTrap, HisTrap, MultiTrap, MidiTrap, MiniTrap, Sephadex, and SpinTrap are trademarks of General Electric Company. © 2017 General Electric Company. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. For local office contact information, visit gelifesciences.com/contact gelifesciences.com GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden KA1392071117PP 23

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