Buffers & Reagents

NEW! BioLock - Biotin Blocking Solution

Biotin is often present in cell culture media. Its nearly irreversible binding to Strep-Tactin has to be taken into account for two reasons when recombinant proteins secreted to the cell culture supernatant are aimed to be directly purified via Strep-Tactin affinity chromatography:

It efficiently competes with the (Twin-)Strep-tag target proteinthereby preventing it from getting bound to the resin and,

moreover,

it inactivates Strep-Tactin resins and thus make a re-use impossible.

BioLock is a ready-to-use and cost effective solution for the removal of free biotin from cell culture supernatant. It simply has to be added to the cell culture supernatant prior to purification.

The required amount of BioLock for effective biotin blocking can easily be determined since 1ml BioLock blocks 70 μg biotin. In addition we provide a table with the required BioLock amounts for standard mammalian and insect cell culture media at
www.iba-lifesciences.com/technical-support.html >> troubleshooting>> Biotin Blocking

Anhydrotetracycline

For induction of tet promoter controlled pASK-IBA vectors. The E. coli expression cassette of the Strep-tag®/Strep-Tactin® overexpression system is under transcriptional control of the tetA promoter/operator and repressor. The promoter is induced by a low concentration of anhydrotetracycline (AHT) saving costs and minimizing the antibacterial influence of AHT.

IBA-lyse Bacterial Lysis Buffer

The new IBA-lyse Bacterial Lysis Buffer is optimized for preparation of a cleared lysate after cytoplasmic expression of Strep-tag® fusion proteins.

The buffer is formulated for gentle disruption of E. coli and release of proteins. It consists of a reagent mix compatible with subsequent Strep-tag® purification. The protocol provides a simple, rapid, inexpensive and most importantly more reproducible and milder alternative to mechanical methods such as sonication or French Press for preparation of cleared lysates to be submitted to affinity chromatography. In addition, it avoids unwanted heating which occurs with e.g. sonication. Avidin, which is included in the buffer, blocks biotin contaminations which could damage the Strep-Tactin® purification columns or cause background on Western blots.

The IBA-lyse buffer comprises:

• Buffer B which contains 1 mM EDTA, Tween 20 and avidin
• Lyophilized lysozyme
• Lysozyme reconstitution buffer
• Lyophilized DNase I
• DNase reconstitution buffer

Buffer L - Lysis buffer for mammalian cell preparation

Lysis buffer for preparation of cell extracts of mammalian cells.

Strep-tag® Washing Buffer (Buffer W)

For removal of unbound material during Strep-tag® protein purification via Strep-Tactin® columns.

Desthiobiotin: Reagent for Strep-tag® II Elution (Buffer E)

Desthiobiotin displaces Strep-tag® II proteins at the biotin-binding site of Strep-Tactin® in a competitive manner resulting in a mild elution of the protein. For your convenience, we offer desthiobiotin ready-to-use in solution (Buffer E). If you desire to prepare your own elution buffer, desthiobiotin is offered as lyophilized powder as well. Please note that after elution with Buffer E the Strep-Tactin® column can be regenerated.

Strep-tag® Regeneration Buffer with HABA (Buffer R)

Contains HABA (2-[4'-hydroxy-benzeneazo]benzoic acid) which displaces desthiobiotin at the biotin-binding site regenerating Strep-Tactin®. The yellow HABA solution turns red when desthiobiotin has been removed from Strep-Tactin® clearly indicating the regeneration process and activity status of the column.

Strep-tag® Protein Purification Buffer Set

Convenient set for the purification of recombinant Strep-tag® fusion proteins on immobilized Strep-Tactin® and for the regeneration of the columns. Consists of Buffers W / E / R.

Buffer BE - Biotin Elution Buffer (MagStrep beads; Spin columns)

Elution buffer for competitive displacement of purified recombinant Strep-tag® or Twin-Strep-tag® ( formerly known as One-STrEP-tag) fusion proteins from immobilized Strep-Tactin®. The buffer is recommended for the elution of MagStrep beads and Strep-Tactin® Spin columns.
This buffer is also recommended for use with Twin-Strep-tag® in protein complex or PPI applications since it contains Biotin which has a higher affinity to Strep-Tactin® and therefore more efficiently competes with the (Twin)Strep-tag® binding. The regeneration of the Strep-Tactin® columns in PPI applications is not recommended to avoid the isolation of cross contaminations.

Please note that after elution with Buffer BE the resin cannot be regenerated.

Avidin

Prevents biotin and biotinylated proteins from binding to Strep-Tactin® columns during Strep-tag® protein purification. This is important if growth media are used containing free Biotin (for further information see “Biotin Blocking”).

Biotin Blocking Buffer (Western blots)

For blocking biotinylated proteins in Western blots (like BCCP protein from E. coli, see Applications), the membrane is incubated with Biotin Blocking Buffer. Use a dilution of 1:1000 in standard Western blot blocking reagent prior to detecting Strep-tag® proteins with Strep-Tactin® conjugates.

Strep-tag II Peptide

For mild elution of Strep-tag® proteins from StrepMAB-Classic Macroprep® columns.

it inactivates Strep-Tactin resins and thus make a re-use impossible.

BioLock is a ready-to-use and cost effective solution for the removal of free biotin from cell culture supernatant. It simply has to be added to the cell culture supernatant prior to purification.

The required amount of BioLock for effective biotin blocking can easily be determined since 1ml BioLock blocks 70 μg biotin. In addition we provide a table with the required BioLock amounts for standard mammalian and insect cell culture media at
www.iba-lifesciences.com/technical-support.html >> troubleshooting>> Biotin Blocking

Anhydrotetracycline

For induction of tet promoter controlled pASK-IBA vectors. The E. coli expression cassette of the Strep-tag®/Strep-Tactin® overexpression system is under transcriptional control of the tetA promoter/operator and repressor. The promoter is induced by a low concentration of anhydrotetracycline (AHT) saving costs and minimizing the antibacterial influence of AHT.

IBA-lyse Bacterial Lysis Buffer

The new IBA-lyse Bacterial Lysis Buffer is optimized for preparation of a cleared lysate after cytoplasmic expression of Strep-tag® fusion proteins.

The buffer is formulated for gentle disruption of E. coli and release of proteins. It consists of a reagent mix compatible with subsequent Strep-tag® purification. The protocol provides a simple, rapid, inexpensive and most importantly more reproducible and milder alternative to mechanical methods such as sonication or French Press for preparation of cleared lysates to be submitted to affinity chromatography. In addition, it avoids unwanted heating which occurs with e.g. sonication. Avidin, which is included in the buffer, blocks biotin contaminations which could damage the Strep-Tactin® purification columns or cause background on Western blots.

The IBA-lyse buffer comprises:

• Buffer B which contains 1 mM EDTA, Tween 20 and avidin
• Lyophilized lysozyme
• Lysozyme reconstitution buffer
• Lyophilized DNase I
• DNase reconstitution buffer

Buffer L - Lysis buffer for mammalian cell preparation

Lysis buffer for preparation of cell extracts of mammalian cells.

Strep-tag® Washing Buffer (Buffer W)

For removal of unbound material during Strep-tag® protein purification via Strep-Tactin® columns.

Desthiobiotin: Reagent for Strep-tag® II Elution (Buffer E)

Desthiobiotin displaces Strep-tag® II proteins at the biotin-binding site of Strep-Tactin® in a competitive manner resulting in a mild elution of the protein. For your convenience, we offer desthiobiotin ready-to-use in solution (Buffer E). If you desire to prepare your own elution buffer, desthiobiotin is offered as lyophilized powder as well. Please note that after elution with Buffer E the Strep-Tactin® column can be regenerated.

Strep-tag® Regeneration Buffer with HABA (Buffer R)

Contains HABA (2-[4'-hydroxy-benzeneazo]benzoic acid) which displaces desthiobiotin at the biotin-binding site regenerating Strep-Tactin®. The yellow HABA solution turns red when desthiobiotin has been removed from Strep-Tactin® clearly indicating the regeneration process and activity status of the column.

Strep-tag® Protein Purification Buffer Set

Convenient set for the purification of recombinant Strep-tag® fusion proteins on immobilized Strep-Tactin® and for the regeneration of the columns. Consists of Buffers W / E / R.

Buffer BE - Biotin Elution Buffer (MagStrep beads; Spin columns)

Elution buffer for competitive displacement of purified recombinant Strep-tag® or Twin-Strep-tag® ( formerly known as One-STrEP-tag) fusion proteins from immobilized Strep-Tactin®. The buffer is recommended for the elution of MagStrep beads and Strep-Tactin® Spin columns.
This buffer is also recommended for use with Twin-Strep-tag® in protein complex or PPI applications since it contains Biotin which has a higher affinity to Strep-Tactin® and therefore more efficiently competes with the (Twin)Strep-tag® binding. The regeneration of the Strep-Tactin® columns in PPI applications is not recommended to avoid the isolation of cross contaminations.

Please note that after elution with Buffer BE the resin cannot be regenerated.

Avidin

Prevents biotin and biotinylated proteins from binding to Strep-Tactin® columns during Strep-tag® protein purification. This is important if growth media are used containing free Biotin (for further information see “Biotin Blocking”).

Biotin Blocking Buffer (Western blots)

For blocking biotinylated proteins in Western blots (like BCCP protein from E. coli, see Applications), the membrane is incubated with Biotin Blocking Buffer. Use a dilution of 1:1000 in standard Western blot blocking reagent prior to detecting Strep-tag® proteins with Strep-Tactin® conjugates.

Strep-tag II Peptide

For mild elution of Strep-tag® proteins from StrepMAB-Classic Macroprep® columns.