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Protein purification product formats 

Protein purification via affinity chromatography is based on the binding affinities between different molecules. During purification, target protein tagged with a short peptide binds to the ligand, which is usually coupled to resin, while unwanted proteins without tag do not bind and can be washed away.

The resin can consist of agarose or magnetic beads. Both come in suspension form. Alternatively, agarose beads can also be prepacked in FPLC, gravity or spin columns. Which format works best for your application depends either on the yield you require or on the initial sample volume.

Choosing the product format based on...

Yield

Sample volume

Choosing protein purification product format based on sample volume

These two schemes should help you decide how to choose the right product format for protein purification among the four most popular formats: columns for FPLC/HPLC devices, gravity flow columns, magnetic beads and spin columns. Please note that the recommendation depends on whether you have the required yield or  sample volume as a starting point.

Product formats in details

FPLC/HPLC columns

Large sample volumes can be purified using columns in automated FPLC/HPLC devices such as Äkta®. FPLC columns come with different resin volumes and can be connected in series to increase the binding capacity. They attach to the device via the standard 10-32 connectors.

  • Broad pH compatibility
  • Excellent pressure stability
  • Outstanding target purity
  • 1 & 5 ml column bed volume available

Product advantages:
  • Quick application of large sample volumes
  • Fully automated protein purification
  • Time saving: No waiting between the purification steps, since the device applies the correct volumes of buffer and sample autonomously.
  • Recording of the complete affinity chromatography protocol, which provides information about the purification quality
  • Reproducible purification results due to the program settings that are set up beforehand and not dependent on the user

Strep-Tactin®XT 4Flow® FPLC column
Content: 1 ml
FPLC column to purify Strep-Tag®II or Twin-Strep-tag® fusion proteins
Strep-Tactin®XT 4Flow® high capacity FPLC column
Content: 1 ml
FPLC column to purify Strep-tag®II or Twin-Strep-tag® fusion proteins
Content: 5 ml
Strep-Tactin® 4Flow® high capacity FPLC column
Content: 5 ml
FPLC column to purify Strep-tag®II or Twin-Strep-tag® fusion proteins - New product! Receive 10% introductory discount* until 31.12.24.
affinity chromatography columns for äkta
Thumbnail for connecting fplc column to an akta device video

Gravity flow columns

Gravity flow columns are ideal for protein purifications with a medium yield. The resin suspensions allow you to pack your own columns according to your need, but you can also choose from other formats, like pre-packed columns.

  • Optimized for column affinity chromatography
  • Variety of different product sizes and matrices

Columns for protein purification

Prepacked columns

Content: 5 ml
Strep-Tactin® Superflow® high capacity column
Content: 5 ml
Gravity flow column to purify Strep-tag®II or Twin-Strep-tag® fusion proteins
Strep-Tactin®XT 4Flow® column
Content: 5 x 0.2 ml
Gravity flow column to purify Strep-tag®II or Twin-Strep-tag® fusion proteins
Strep-Tactin®XT 4Flow® high capacity column
Content: 5 x 0.2 ml
Gravity flow column to purify Strep-tag®II or Twin-Strep-tag® fusion proteins

Suspension

Strep-Tactin® MacroPrep® resin
50% suspension to purify Strep-tag®II or Twin-Strep-tag® fusion proteins
Strep-Tactin®XT 4Flow® high capacity resin
Content: 4 ml
50% suspension to purify Strep-tag®II or Twin-Strep-tag® fusion proteins
Content: 50 ml
Strep-Tactin®XT 4Flow® resin
Content: 50 ml
50% suspension to purify Strep-tag®II or Twin-Strep-tag® fusion proteins
Strep-Tactin® 4Flow® high capacity resin
Content: 4 ml
50% suspension to purify Strep-tag®II or Twin-Strep-tag® fusion proteins - New product! Receive 10% introductory discount* until 31.12.24.

Magnetic beads

Magnetic beads can be used for batch purifications in tubes or 96-well plates to purify proteins in pull-downs, IPs and Co-IPs. The beads can be easily fixed to the reaction tube with a magnet, so you can skip centrifugation without having to worry about losing your sample during pipetting steps.

  • No centrifugation
  • Separation via magnetic separator
  • 2 ml and 10 ml volume available


Pull-down assay magnetic beads
MagStrep® Strep-Tactin®XT beads
Content: 2 ml
Magnetic beads (5% suspension) to purify Strep-tag®II or Twin-Strep-tag® fusion proteins
MagStrep® Strep-Tactin® beads
Content: 2 ml
Magnetic beads (5% suspension) to purify biotinylated proteins

Spin columns

If you need to process multiple small samples in parallel, you can use spin columns with the resin of your choice. They allow you to do a batch purification of a different protein in every tube with a simple setup. 

  • Compatible with conventional 1.5 ml or 2.0 ml reaction tubes
  • Buy a ready-to-use kit or fill empty columns with resin of your choice


Spin columns for protein purification
Strep-Tactin®XT 4Flow® high capacity Spin Column Kit
For rapid purification of Strep-tag®II and Twin-Strep-tag® fusion proteins
Empty Spin Columns
For rapid protein purification with Strep-Tactin®XT 4Flow® high capacity

Resin reuse

Strep-Tactin® and Strep-Tactin®XT resins for protein purification can be regenerated and reused >10 times under ideal conditions without loss in performance. The proper regeneration of the column and resin activation can easily be checked with HABA. The yellow HABA solution turns red (Strep-Tactin®) or orange (Strep-Tactin®XT) upon binding to the engineered biotin binding pockets of Strep-Tactin® and Strep-Tactin®XT clearly indicating that the resin is fully regenerated. Afterwards, HABA can be removed by washing with 1x Buffer W. Once the red color has disappeared the column can be reused. If the biotin binding pocket is blocked or damaged no color shift occurs and the resin cannot be reused.

Resin specifications


 

Strep-Tactin® 

Strep-Tactin®XT

Matrix Superflow®  Sepharose® MacroPrep® 4Flow® MagStrep Strep-Tactin®XT beads
      (only available as 20 ml slurry)    
Binding capacity*/
Dynamic Binding capacity**
classic: 3 mg/ml resin* classic: 3 mg/ml resin* classic: 3 mg/ml resin* classic: 5 mg/ml resin** classic: 25.5 µg/µl resin*
  high capacity: 7.0 mg/ml resin**     high capacity: 14 mg/ml resin**  
Bead structure 6% agarose, crosslinked 4% agarose, crosslinked polymethacrylate 4% agarose, highly crosslinked magnetic core covered with 6% agarose, crosslinked
Bead size 60-160 µm, spherical 45-165 µm 50 µm 50-150 µm, spherical 30 μm (average), spherical
Exclusion limit 6 x 10Da ~3 x 107 Da 1 x 106 Da 3 x 107 Da not specified 
Recommended  flow rate 0.5-1 ml/min  gravity flow 0.5-1 ml/min  0.5-1 ml/min batch purification
pH range for protein binding 7-8 7-8 7-8 4-10 6-10
Max pressure 9.6 bar gravity flow 70 bar 3.5 bar batch purification
Storage 4 °C, do not freeze 4 °C, do not freeze 4 °C, do not freeze 4 °C, do not freeze 4 °C, do not freeze
Shipment RT RT RT RT RT
Eluent desthiobiotin
desthiobiotin
desthiobiotin
biotin biotin
Regeneration buffer Buffer R Buffer R Buffer R Buffer XT-R
0.1 M NaOH
0.1 M NaOH
Features and recommendations
  • Strep-Tactin® Superflow high capacity binds three to five times more protein in comparison to Strep-Tactin® Superflow
  • Can be used for the purification of biotinylated proteins
  • For average sized proteins (<90 kDa)
  • For proteins that are hard to elute from Strep-Tactin®XT resins, like proteins with multiple tags or multimers
  • Suitable for use in FPLC columns
  • For large proteins (>90 kDa)
  • Can be used for the purification of biotinylated proteins
  • For proteins that are hard to elute from Strep-Tactin®XT resins, like proteins with multiple tags ore multimers
  • Only suitable for gravity flow due to low pressure stability
  • Synthetic support
  • Reduced unspecific binding
  • Strep-Tactin®XT 4Flow® high capacity binds three to fove times more target protein compared to Strep-Tactin®XT 4Flow®
  • Applicable for all protein types, especially large (>90 kDa), low abundant and challenging proteins
  • Strep-Tactin®XT 4Flow is recommended for first time users of the Strep-tag® system
  • Optimized for Batch purification
  • Beads can be separated with a magnetic device, no centrifugation necessary
  • Suitable for purification in 96-well plates

* Max binding capacity for a Strep-tag® protein (30kDa).

** Dynamic binding capacity (DBC) was determined with mCherry-Twin-Strep-tag (30 kDa) under realistic operation conditions and shows the amount of protein which is bound until 10% of the protein is found in the flow through.

Resin specifications


Ligand Strep-Tactin®  Strep-Tactin®XT
Matrix 4Flow® high capacity 4Flow® 4Flow® high capacity MagStrep® Strep-Tactin®XT beads
Binding capacity 20 mg/ml resin* 11 mg/ml resin* 31 mg/ml resin* 25.5 µg/µl resin**
Bead structure 4% agarose, highly crosslinked 4% agarose, highly crosslinked 4% agarose, highly crosslinked magnetic core covered with 6% agarose, crosslinked
Bead size 50-150 µm, spherical 50-150 µm, spherical 50-150 µm, spherical 30 μm (average), spherical
Exclusion limit 3 x 107 Da 3 x 107 Da 3 x 107 Da not specified 
Recommended  formats Gravity flow, FPLC Gravity Flow, FPLC Gravity Flow, FPLC Batch purification
pH range for protein binding 7-8 4-10 4-10 6-10
Max pressure 3.5 bar 3.5 bar 3.5 bar  
Storage 2-8 °C, do not freeze 2-8 °C, do not freeze 2-8 °C, do not freeze 2-8 °C, do not freeze
Shipping RT RT RT RT
Eluent desthiobiotin
 
biotin biotin biotin
Regeneration buffer 100 mM NaOH Buffer XT-R
100 mM NaOH
 
Buffer XT-R
100 mM NaOH
 
100 mM NaOH
Activity Test Buffer R (HABA) Buffer R (HABA) Buffer R (HABA)  
Features and recommendations
  • Strep-Tactin® 4Flow® high capacity combines a low percentage agarose and pressure stability
  • For proteins that are hard to elute from Strep-Tactin®XT resins, like proteins with multiple tags or multimers
  • Applicable for proteins of all types and sizes
  • Strep-Tactin®XT 4Flow® is recommended for first time users of the Strep-tag® system 
  • Applicable for all protein types and sizes, especially low abundant and challenging proteins
  • Strep-Tactin®XT 4Flow® high capacity binds three to five times more target protein compared to Strep-Tactin®XT 4Flow®
  • Recommended for the purification of well-expressed proteins or in large-scale purification
  • Optimized for Batch purification
  • Beads can be separated with a magnetic device, no centrifugation necessary
  • Suitable for high-throughput purification in 96-well plates

*determined with a 52 kDa Twin-Strep-tag® fusion protein

**determined with a 30 kDa Twin-Strep-tag® fusion protein

Application example

Cell culture supernatant of MEXi-293E cells (24 ml) was spiked with 240 µg of a large protein (130 kDa) C-terminally tagged with the Twin-Strep-tag®. Protein purification was carried out with a 0.2 ml Strep-Tactin®XT 4Flow® high capacity gravity flow column. After sample application the column was washed with 1x Buffer W. The Twin-Strep-tag® protein was eluted by three elution steps with 0.7, 1.5 and 0.8 CV 1x Buffer BXT. Purification results were analyzed by SDS-PAGE (A) and western blot (B). Both show a molecular weight marker (M*, Precision Plus Protein™ Unstained Protein Standards, Strep-tagged recombinant (BioRad), M** PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific)), the spiked cell supernatant (S), flow through (FT), the 1st and 5th wash fraction (W1 and W5, respectively) and the elution fractions (E1-E3). Strep-Tactin® HRP was applied (1:4000) for detection via western blot.