CD3+ cells were isolated from buffy coats (A, n=10) or murine splenocytes (B, n=7) using the Fab-TACS® approach for affinity chromatographic cell isolation. Data are displayed as mean ± SEM. On average, around 85% of CD3+ cells present in the starting material of both human and murine samples were retrieved after CD3-specific enrichment.

CD3+ cells were isolated from buffy coats (A, n=10) or murine splenocytes (B, n=7) using the Fab-TACS® approach for affinity chromatographic cell isolation. Data are displayed as mean ± SEM. On average, 99% of cells were viable after isolation from buffy coats and 91% after enrichment from murine splenocytes.

CD3+, CD4+ or CD8+ T cells were isolated from PBMCs using Fab-Streps and Strep-Tactin® Magnetic Microbeads according to the Fab-TACS® approach for magnetic cell isolation. Dead cells were determined using DAPI staining. Doublet and debris discrimination were performed using different FSC/SSC signals. > 93% of cells were viable after all isolation procedures.

Human CD3+ T cells were isolated with CD3 Fab-Streps and Strep-Tactin® Magnetic Microbeads according to the Fab-TACS® approach for magnetic cell isolation. Cells were stained with CFSE and cultured with CD3/CD28 Streptamers in a 48 well plate for 5 days. Proliferation was measured in pregated CD4+ (upper panel) or CD8+ T cells (lower panel) using Flow Cytometry. Dead cells were excluded from the analysis using PI.

Human CD3+ T cells were isolated with CD3 Fab-Streps and Strep-Tactin® Magnetic Microbeads according to the Fab-TACS® approach for magnetic cell isolation. Cells were cultured for 13 days with CD3/CD28 Streptamers for cell expansion. Fold expansion was measured.

Human CD3+ T cells were isolated with CD3 Fab-Streps and Strep-Tactin® Magnetic Microbeads according to the Fab-TACS® approach for magnetic cell isolation. Cells were cultured with (A) or without (B) CD3/CD28 Streptamers for T cell expansion. Cluster formation was evaluated using microscopy.

Type: antibody

Yield: 96 mg/l (represented by heavy (HC) and light chain (LC))

The monoclonal rat antibody (mAb) was cloned into the pDSG-IBA102 vector in order to fuse the heavy chain (HC) C-terminally with the Twin-Strep-tag® and the BM40 secretion signal. The transfection of MEXi 293E cells was performed in MEXi-TM transfection medium (250 ml) with  polyethylenimine (PEI, 25 kDa). The cells were incubated for 4 hours (37 °C, 5% CO2, 125 rpm) and when a cell density of 3 x 106 cells/ml was reached, 250ml MEXi-CM culture medium was added. Afterwards, the culture was shifted to 32 °C and incubated until day 10.

In order to divide the cells from the supernatant, the cell suspension was centrifuged according to the MEXi manual. Free biotin was blocked by BioLock containing avidin. The supernatant was used for protein purification via a gravity flow Strep-Tactin® Superflow® high capacity column. WET FRED was used to facilitate loading of the large supernatant volume onto the column.

Yield: 318 mg/l

The coding sequence of the customer protein was cloned into pDSG-IBA102 leading to a recombinant protein with C-terminal Twin-Strep-tag® and BM40 secretion signal. 1050 ml of MEXi-TM was inoculated with MEXi 293E cells. Subsequently, plasmid DNA was added followed by addition of 25 kDa linear PEI. The cells were incubated for 4 hours in MEXi-TM medium at 37 °C, 5% CO2, and 125 rpm in an orbital shaking incubator. Cells were diluted to 7.5 x 105 cells/ml by addition of one volume MEXi-CM and kept at 37 °C for 7 days. Afterwards, cells were pelleted and the supernatant containing the customer protein was harvested. The customer protein was finally purified using Strep-Tactin®XT Superflow®. The figure shows the elution fractions (E1-E3). A dilution of 1:10 was prepared for E1 and E2 for SDS-PAGE analysis.

Important exosome sources also include a variety of human bodily fluids such as blood and urine. Similar to isolations from cell culture supernatants, 96% of particles retrieved by exosome isolation from serum of humans exhibited the typical exosome size of 30 to 150 nm (E).

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.

Coding sequences of virus-like particles (VLPs, 4.8 MDa) were cloned into pASK-IBA7 to obtain proteins localized to the cytoplasm and N-terminally tagged with Strep-tag®II. The plasmid was transformed into E. coli and the cell culture (1 l) was induced at OD600 of 0.6 by addition of anhydrotetracycline (AHT). Protein expression was performed at 37 °C for 3 h at 200 rpm. Afterwards, cells were harvested, resuspended with 20 ml 1x Buffer W (100 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA), and sonicated. The insoluble material was pelleted, and the lysate was applied to a Strep-Tactin® Superflow® cartridge (1 ml/min flow rate). After washing, the target protein was eluted with 2.5 mM desthiobiotin. Purification results were analyzed by SDS-PAGE. Marker (M) and two different elution fractions of VLPs fused to Strep-tag®II (lane 1 and 2) are shown.

Data kindly provided by L. Stöckl and B. Brandenburg, Robert-Koch-Institute, Berlin. Brandenburg, et al. (2005): A Novel System for Efficient Gene Transfer Into Primary Human Hepatocytes Via Cell-Permeable Hepatitis B Virus–like Particle, HEPATOLOGY (42), 1300-1309.

Five different proteins were purified with Strep-Tactin® Superflow® and Strep-Tactin®XT Superflow® to compare the yield with both rein types. Here, we found that Strep-Tactin®XT Superflow® allows the purification of on average a 2-fold higher protein yield than Strep-Tactin® Superflow®.

An advantage of Strep-Tactin®XT compared to Strep-Tactin® is that it allows intensive washing without loss in protein yield. To illustrate this, mCherry-Twin-Strep-tag from E. coli supernatant was purified with a gravity flow column either containing 1 ml Strep-Tactin® Superflow® or Strep-Tactin®XT Superflow®. During purification, the columns were washed with 8 column volumes of 1x Buffer W to reach a constant absorption ratio at A260/280 nm. Then, the target protein was eluted with 50 mM biotin. Samples of the last wash fractions (wash 5-8) and the elution fraction were analyzed by SDS-PAGE. In comparison to Strep-Tactin® Superflow®, Strep-Tactin®XT Superflow allows intensive washing without loss of protein yield. The high affinity (pM range) between Strep-Tactin®XT and Twin-Strep-tag® leads to a tighter binding and prevents a premature elution of the target protein during the wash steps. The elution fractions were further analyzed by Western Blot and detected with Strep-Tactin®HRP, showing that the further bands are alternative variants of mCherry-Twin-Strep-tag.

For some recombinant proteins, for example from plants, purification results of Strep-Tactin® MarcoPrep® can differ from those of Strep-Tactin® Sepharose®, due to different resin properties. The SDS-PAGE gel shows the purification of a recombinant protein which exhibits exceptionally high non-specific protein binding. In this case, contaminants were almost completely removed by application of Strep-Tactin® MacroPrep®, whereas the contaminants co-eluted with the recombinant protein after the same purification protocol using Strep-Tactin® Sepharose®. Please note, normally behaving recombinant proteins can be purified in a single step to homogeneity using Strep-Tactin® Sepharose®, however, the example shows that it may be reasonable to change the resin if non-specific protein binding occurs.

Dynamic binding capacity of Protein A remains stable after treatment with 0.5 M NaOH over~100 cycles  (1 cycle = 15 min).

The membrane protein CB2 was fused with Twin-Strep-tag® and then immobilized on a CM4 sensor chip coated with Strep-Tactin®XT.

Used conditions:

50 mM Tris pH 7.5, 100 mM NaCl
10 % glycerol, 0.1 % DDM, 0.5 % CHAPS, 0.1 % CHS

Data was published in A. Yeliseev et al., Protein Expression and Purification 131 (2017) 109-118

Three different regeneration conditions were tested on Strep-Tactin®XT CM5 sensorchips:

10 mM NaOH / 500 mM NaCl
3 M MgCl2
3 M GuHCl

for three different Twin-Strep-tag® fusion proteins:

mCherry-Twin-Strep-tag
GAPDH-Twin-Strep-tag
mAb-Twin-Strep-tag

We recommend the application of 3 M GuHCl for optimal regeneration conditions, since this buffer led to the best regeneration results for all test proteins. However, depending on the target protein, other buffers are applicable as well.

Strep-Tactin® coated microplate was incubated with recombinant H. pylori urease tagged with Strep-tag® II, followed by three washing cycles. Afterwards, the microplate was incubated with human sera followed by three washing cycles. Third incubation occurred with rabbit anti-human lgG conjugated to horseradish peroxidase (HRP), followed by 3 washing cycles. Amount of bound antibodies from human sera was determined by colorimetric reaction of HRP.

Application: Western Blot

1-1000 ng/lane of GFP C-terminally tagged with Strep-tag®II (28 kDa) were separated by SDS-PAGE and blotted onto a membrane. Detection occurred with Strep-Tactin® AP.

Application: Dot Blot

0.5-500 ng/spot of GFP-Strep-tagII (28 kDa) or alkaline phosphatase C-terminally tagged with Strep-tag®II (monomer 48.5 kDa) were spotted onto a membrane. Detection occurred with Strep-Tactin® AP.

Application: Confocal microscopy (Immunofluorescence)

Dilution: 1:500

HeLa cells were grown on glass coverslips, fixed (4% PFA/PBS), quenched (15 mM glycine/PBS), permeabilized (0.1% saponin/PBS), blocked (1% BSA, 0.01% saponin in PBS), and stained with StrepMAB-Classic (1:500) to confirm the expression of SARS-CoV-2 ORF3a-Twin-Strep-tag. Anti-mouse 488 was used as secondary antibody. Nuclei were stained with DAPI and the imaging occurred with a LSM700 confocal microscope (63×/1.4 NA oil immersion objective; ZEISS).

Source: Data kindly provided from James Edgar, Department of Pathology at the University of Cambridge.

Antigen-specific cells were visualized with Strep-Tactin® PE coupled to MHC I-Strep H2-Kd specific for LLO91-99 . After addition of 1 mM d-biotin, subsequent dissociation of monomerized MHC molecules was monitored at different time points as indicated. 20 minutes after biotin addition, staining reagents were largely removed from the cells. Staining and dissociation were performed at 4 °C.

Used reagents:

6-7014-001 MHC I-Strep H2-Kd, LLO91-99
6-5000-001 Strep-Tactin® PE

Staining of Her2/neu-specifc CD8+ T cells (kindly provided by H. Bernhard, TU Munich) with HLA-A*0201/Her2/neu369-377 Streptamers® as compared to conventional multimers (left diagram). Complete dissociation of Streptamer® staining reagents from the cells is shown after a brief incubation (30 min) in the presence of 1 mM d-biotin (right diagram). Antigen-specific T cells were visualized with Strep-Tactin® PE coupled to MHC I-Strep HLA-A*0201 specific for Her2/neu369-377. Staining and dissociation were performed at 4 °C.

Used reagents:

6-7008-001 MHC I-Strep HLA-A*0201, Her2/neu369-377
6-5000-001 Strep-Tactin® PE