Frequently asked questions

Cloning and gene expression

Strep-tag purification system

16. Should columns or magnetic beads be used?
    Generally affinity column purification is recommended. Batch purification (with Strep-Tactin^® coated magnetic beads) is only recommended if the expression rate of the recombinant protein is above 1 mg per litre E. coli culture. Lower expression rates are possible using the One-StrEP-tag or the Strep-Tactin Superflow high capacity matrix.
    
    
17. What degree of purity can be expected?
    Over 95 %. It must be mentioned that potential impurities may result from non-specific interactions of the recombinant protein itself with other proteins leading to lower purity grades. More purity related FAQs you find in chapter "Contaminations".
    
    
18. How can the protein be eluted?
    Elution is performed by the addition of 2.5 mM desthiobiotin, a derivative of biotin. It is a stable, reversibly binding (columns can be regenerated) low molecular weight substance which does not interfere with the protein or general protein assays.
    
    
19. Is the protein's bioactivity preserved?
    Yes. Bioactivity is preserved due to the mild washing and elution conditions by the use of physiological buffers.
    
    
20. When the protein elutes from the column, is it complexed with desthiobiotin?
    No. Desthiobiotin can be removed via gel filtration or dialysis
    
    
21. Is the Strep-tag^® system stable in the presence of imidazole?
    Yes, the Strep-tag system tolerates up to 250 mM imidazole in the protein extract. Therefore, elution fractions from Ni-NTA resins can be directly applied on Strep-Tactin columns, which is very useful in double-tag purification protocols.
    

Purification columns & cartridges

22. What is the difference between columns and cartridges?
    Gravity flow columns with Strep-Tactin Sepharose [Cat.-no.: 2-1202-001], Strep-Tactin Superflow [Cat.-no.: 2-1207-001] or Strep-Tactin MacroPrep [Cat.-no.: 2-1506-001] resins are designed for gravity flow chromatography.
    Pre-packed columns with Strep-Tactin POROS resin [Cat.-no.: 2-1203-017, 2-1205-017] are designed for high linear flow rates.
    The new H-PR cartridges ("highly pressure-resistant") with Strep-Tactin Superflow [Cat.-no.: 2-1231-001, 2-1232-001], Strep-Tactin MacroPrep [Cat.-no.: 2-1532-001, 2-1511-001] or Ni-NTA Superflow [Cat.-no.: 2-3231-001, 2-3232-001] resins are designed for automated chromatography workstations with 10-32 connections such as ÄKTATM. The H-PR cartridges are highly pressure resistant for use in FPLC/HPLC, can tolerate 20 bar and can also be used with a flow restrictor.
    Our older version of Luer-lock cartridges, which were also designed for FPLC and HPLC, is about to be discontinued.
    
    
23. Which adapters are necessary using H-PR cartridges?
    For HPLC and ÄKTA systems no adapters are needed. For chromatography workstations (or syringes or peristaltic pumps) other than HPLC and ÄKTA, adapters are required [Cat.-no.: 2-1022-001, 2-1023-001, 2-1025-001, 2-1026-001]. To connect several H-PR cartrigdes in series to enlarge capacity please use coupling adapters [Cat.-no.: 2-1026-001]. For the use of H-PR cartridges with the older Luer-lock system please use syringe adapters [Cat.-no.: 2-1021-001].
    
    
24. What kind of matrix should be used?
    The best support for the purification depends on the recombinant protein and is normally not predictable. For rapid optimization of your purification results, we are offering two column evaluation sets [Cat.-no.: 2-1001-001, 2-1001-011] with Strep-Tactin immobilized to the supports Sepharose^®, Superflow^® or MacroPrep^®.
    
    
25. What is the recommended linear flow rate* for Strep-tag^® purification?
    
    

    Sepharose	Superflow	MacroPrep	POROS
    gravity flow	Up to 300cm/h	Up to 300 cm/h	300 – 500 cm/h

    *Volumetric flow rate (ml/min) = Linear flow rate (cm/h) divided by 60 and multiplied by the column cross sectional area (cm²)
    
    
26. Which volumes should be used?
    
    

    Column bed volume:	Protein extract volume:	Washing buffer volume :	Elution buffer volume:
    0.2 ml	0.1 – 2 ml	5 x 0.2 ml	6 x 0.1 ml
    1 ml	0.5 – 10 ml	5 x 1 ml	6 x 0.5 ml
    5 ml	2.5 – 50 ml	5 x 5 ml	6 x 2.5 ml
    10 ml	5 – 100 ml	5 x 10 ml	6 x 5 ml

    
    
    
27. Why is the colour of the MacroPrep^® Strep-Tactin column not intense red after regeneration with HABA?
    MacroPrep is not as transparent as Sepharose^® or Superflow^®. Thus, the colour shift is to some kind of pink only
    
    
28. How should the columns be stored?
    4 °C, overlayed with buffer W or, for shorter periods, with buffer R.
    
    
    

Alternate buffer conditions

29. Can the column be treated with chaotropic salts?
    Yes. 1 column volume of 6 M guanidine/HCl can be applied for removing aggregates which may have formed after several purification runs. Do not store the column in the presence of guanidine. Restore immediately native buffer conditions after guanidine treatment by washing with, for example, Tris buffer at pH 8.0 (e.g. Buffer W).
    
    
30. Can detergents or other buffer systems be used?
    Yes. High salt, reducing reagents like mercaptoethanol, chelating reagents, and detergents are allowed but not necessary. However, the pH has to be above pH 7.0.
    
    
31. Which reagents are compatible with the Strep-tag^®/Strep-Tactin^® interaction?
    
    

    Reagent	Concentration
    Reduction Agents
    DTT
    β-mercaptoethanol
    Non-Ionic Detergents
    C8E4; Octyltetraoxyethylene	max. 0.88 %
    C10E5; Decylpentaoxyethylene	0.12 %
    C10E6	0.03 %
    C12E8	0.005 %
    C12E9; Dodecyl nonaoxyethylene (Thesit)	0.023 %
    DM; Decyl-β-D-maltoside	0.35 %
    LM; N-dodecyl-β-D-maltoside	0.007 %
    NG; N-nonyl-β-D-glucopyranoside	0.2 %
    OG; N-octyl-β-D-glucopyranoside	2.34 %
    TX; Triton X-100	2 %
    Tween 20	2 %
    Ionic Detergents
    N-lauryl-sarcosine	2 %
    8-HESO;N-octyl-2-hydroxy-ethylsulfoxide	1.32 %
    SDS; Sodium-N-dodecyl sulfate	0.1 %
    Zwitter-Ionic Detergents
    CHAPS	0.1 %
    DDAO; N-decyl-N,N-dimethylamine-N-oxide	0.034 %
    LDAO; N-dodecyl-N,N-dimethylamine-N-oxide	0.13 %
    Others
    Others
    Ammonium sulfate (NH4)2SO4	2 M
    CaCl2	max. 1 M
    EDTA	50 mM
    Ethanol	10 %
    Guanidine	max. 1 M
    Glycerol	max. 25 %
    Imidazole	max. 250 mM
    MgCl2	1 M
    NaCl	5 M
    Urea	max. 1 M

    
    Note: These reagents have been successfully tested for the purification of e.g. GAPDH-Strep-tag with concentrations up to those mentioned. For reagents not marked with “max.” higher concentrations may be possible, though. Since binding depends on the sterical accessibility of Strep-tag in the context of the particular protein the possible concentration may deviate from the given value for other proteins.
    
    

Contaminations

32. Is the presence of biotinylated proteins in the host organism in case of proteins expressed in the cytoplasm a problem?
    No. Generally, the cell internal content of biotinylated proteins and free biotin is rather low and not a threat for significant inactivation of the Strep-Tactin resin. Therefore, no avidin has necessarily to be added to the extract when purification is intended.
    Example: An E. coli extract derived from a 1 l culture with OD550 = 1 has a total biotin content of around 1 nmol only (biotin capacity ≅ 350 nmol/ml sedimented resin).
    
    
33. Is the presence of free biotin in the medium (in case of proteins secreted to the medium) a problem (eukaryotic expression)?
    Free Biotin inactivates Strep-Tactin resins (biotin capacity ≅ 350 nmol/ml sedimented resin) and has to be removed or masked prior to affinity chromatography. This is mostly relevant when cell culture supernatant, containing secreted recombinant protein, is directly subjected to Strep-Tactin affinity chromatography because some media for insect cells or mammalian cells contain significant amounts of biotin (see table below).
    The simplest way to get rid of the biotin problem for purification of secreted eukaryotic proteins is irreversible masking by the addition of avidin [Cat.-no.: 2-0204-050] using 120 mg avidin per mg contaminating biotin. This procedure is recommendable at high expression levels.
    
    Biotin contents of standard mammalian cell culture media (1 mg biotin corresponds to 4 µmol):
    
    

    Medium	D-Biotin [mg/l]	Citation
    BME (Eagle)	1	Eagle H. (1965), Proc. Soc. Exp. Med. 89, 362
    CMRL 1066	0.01	Parker, R.C., et al. (1957) Special Publications, N.Y. Academy of Sciences, 5, 303
    DMEM	-	Dulbecco, R. Freeman, G. (1959) Virology 8, 396. Smith, J.D., Vogt, M. and Dulbecco, R. (1960) Virology 12, 185
    DMEM/F-12	0.035
    Hams F10	0.024	Ham, R.G. (1963), Exp. Cell Res., 29, 515
    Hams F12	0.007	Ham, R.G. (1965), Proc. Nat. Acad, Sci., 53, 288
    Fischer's	0.01	Fischer, G.A. and Sartorelli, A.S. (1964), Methods in Med. Res. 10.
    Iscove's (IMDM)	0.013
    Leibovitz's L-15	-	Leibovitz, A. (1963) Am. J. Hyg. 78, 173
    McCoys 5A	0.2
    MCDB 131	0.0073
    Medium 199	0.01	Morgan, Morton and Parker (1950) Proc. Soc. Exp. Biol. Med., 73, 1
    a MEM	0.1
    NTCC 109/135	0.025
    RPMI 1640	0.2	Moore, G.E., Gerner, R.E. and Franklin, H.A. (1967) A.M.A. 199, 519
    Waymouth's MB 752/1	0.02
    Williams' E	0.5	Williams, G.M. and Gunn, J.M. (1974) Exp. Cell. Res., 89, 39

    
    

    Biotin contents of standard insect cell culture media (1 mg biotin corresponds to 4 µmol):

    Medium	D-Biotin [mg/l]	Citation
    Grace's	0.01	Grace, T.C. (1962) Nature 195, 788
    Schneider's	-	Schneider, I. (1964), Exp. Zool. 156, 1 , 91
    SF900II from Invitrogen	0.1	determined by IBA
    SF3 from PromoCell	0.1	determined by IBA
    ExCell 401 from JRH Biosciences	0.1	determined by IBA
    Express Five from Gibco/Invitrogen	0.15	determined by IBA
    Graces from Gibco/Invitrogen	-	determined by IBA
    Insect Xpress from Cambrex	0.1	determined by IBA

    
    
    
34. How can additional bands from biotinylated proteins on Western blots be avoided?
    For blocking biotinylated proteins in Western blots, we offer a Biotin Blocking Buffer containing avidin [Cat.-no.: 2-0501-002]. Use a dilution of 1:1000 in standard Western blot blocking reagent prior to detecting Strep-tag proteins with Strep-Tactin conjugates. Alternatively, monoclonal antibodies against Strep-tag [Cat.-no.: 2-1507-001, 2-1509-001] can be used, which do not recognize biotin.
    
    
35. How can desthiobiotin be separated from the Strep-tag^® protein after elution?
    Either use gel filtration or dialyse the elution fractions against your buffer of choice. Usually this is not necessary, though.
    
    

Regeneration

36. How can the Strep-Tactin^® matrix be regenerated?
    If desthiobiotin is used for elution, the Strep-Tactin^® matrix can be regenerated with an azo dye (HABA) which, when applied in excess, displaces desthiobiotin. The dye is yellow in solution and shifts to red when bound by Strep-Tactin which allows the visual control of the regeneration process and the functional status of the column. As long as a colour gradient between the top and the bottom of the column is visible, it is not fully regenerated.
    Regeneration is not possible, if biotin has been used for elution (Strep-Tactin Spin Columns, Strep-Well HT Purification Plates, MagStrep (Strep-Tactin coated Magnetic Beads), One-StrEP protein:protein interaction analysis) since biotin binds nearly irreversible to Strep-Tactin.
    
    
37. What is the mechanism for HABA displacing desthiobiotin from the Strep-Tactin^® column?
    HABA binds to the same Strep-Tactin binding pocket as Strep-tag (or biotin) does, but with very low affinity only. Nevertheless, it accelerates desthiobiotin removal because it is applied in excess. That means each time when a Strep-Tactin:desthiobiotin complex dissociates on the column, the resulting free Strep-Tactin binding site may be occupied by HABA or desthiobiotin in competition. As HABA is applied in excess, desthiobiotin has only a reduced chance to find a free binding pocket, which finally results in accelerated removal of desthiobiotin from the column.
    
    

Detection / Assay

38. How can the tag be detected?
    For fast detection we offer Strep-Tactin conjugated to HRP or AP. The Strep-tag HRP Detection Kit [Cat.-no.: 2-1502-000] and the Strep-tag AP Detection Kit [Cat.-no.: 2-1503-000] provide the necessary tools to detect Strep-tag II fusion proteins in Western blots. These ready-to-use systems include all buffers and reagents for blocking, washing and the chromogenic reaction. For high specificity the monoclonal antibody against Strep-tag II can be used [Cat.-no.: 2-1507-001, 2-1509-001]. It is available affinity purified and unlabelled or affinity purified and labelled with HRP.
    
    
39. Which detection procedure (ECL or chromogenic) do you recommend for HRP Strep-Tactin® conjugates?
    Both, chemiluminescene as well as chromogenic detection, are possible. Please note, that the application protocols for these procedures differ. The protocols are described in the "Western blot protocol" which is also available as pdf on the IBA download/tech info site.
    
    
40. What are the KD-values of the monoclonal antibodies?
    KD StrepMAB-Classic = approximately 1 µM.
    KD StrepMAB-Immo: approximately 1 pM.
    These antibodies are monoclonal mouse antibodies, type IgG1.
    
    
41. Do the StrepMAB antibodies also work in immunoprecipitation and pull down assays?
    Yes, several applications are possible. We recommend to use the One-STrEP-tag (Strep-tag II is also possible) for the bait protein and to precipitate with Strep-Tactin Superflow or Strep-Tactin magnetic beads using biotin for the elution step. Alternatively, StrepMAB-Classic MacroPrep can be used for precipitation using the Strep-tag peptide for the elution step. Please note, if you have to use classical immunoprecipitation without an elution step, we recommend StrepMAB-Immo MacroPrep for immunoprecipitation because of its nearly irreversible binding to the One-StrEP-tag or Strep-tag II.
    
    
42. Can a tagged protein be detected in situ?
    Yes. It can be detected via EM or FM with streptavidin which is labelled with colloidal gold or FITC.
    
    
43. Has the Strep-tag^® technology been tested in Biacore analysis?
    Yes. StrepMAB-Immo, high-affinity Strep-tag® II specific monoclonal antibody [Cat.-No.: 2-1517-001], is optimal for capturing SerAla-Strep-tag^® II fusion proteins on solid phases such as Biacore chips or microplates.