Protein Technologies – Bioseparation Products
Mimetic Ligand Adsorbents: Mimetic Blue™
MIMETIC BLUE™ SA
DESIGNER LIGAND FOR PURIFICATION/REMOVAL OF SERUM ALBUMIN
- HIGHLY STABLE LIGAND, RESISTANT TO SODIUM HYDROXIDE
- EASY SCALE-UP
This affinity adsorbent has been developed specifically for the
isolation
of serum albumin from different sources. The Mimetic Blue™ SA
ligand consists of a blue chromophore (similar to that of C.I. Reactive
Blue 2) linked to cross-linked beaded agarose by a highly stable
spacer-arm linkage. Unlike C.I. Reactive Blue 2 based adsorbents,
Mimetic Blue™ SA displays a high affinity for a variety of serum
albumins, including bovine serum albumin (BSA). Consequently,
Mimetic Blue™ SA may be used to purify albumin or to effect its removal
from products isolated from serum, plasma or cell culture media.
The ligand is bonded onto 6% cross-linked agarose by a highly stable
bonding method which renders the adsorbent resistant to treatment with
1M NaOH. Consequently, the use of NaOH cleaning procedures
ensures that Mimetic Blue™ SA can be sanitized and used repeatedly for
purification from relatively crude protein extracts.
Scale-up is simple. The adsorbent is manufactured to GMP and ISO 9001:2000 standards and large batch sizes are available for process applications. Drug Master Files (DMF) have been submitted for the facility to make traceability and compliance easier.
Specifications
| Support Matrix: | 6% cross-linked agarose |
| Bead Size: | 45 - 165 microns |
| Exclusion Limit (Globular Proteins): | 4 x 106 Daltons |
| Immobilized Limit (Globular Proteins): | 4.5 +/- 0.5 µmol/g |
| Temperature Range: | 4 - 120 °C |
| pH Range: | 3 - 13 (continuous) 2 - 14 (intermittent) |
| Solvent Compatibility: | Aqueous solutions of alcohols, esters and ketones |
| Maximum Pressure: | 1 bar (15 psi) |
| Chemical Compatibility: | Compatible with most biological buffers, chaotropes, detergents, chelating agents and thiols. Do not expose to strong oxidizing/reducing agents. |
| Albumin Binding Capacity: | Varies depending upon source, sample purity and conditions. Typically >30mg/ml for BSA at pH 6.0 |
| Recommended Preservative: | Ethanol/0.1M NaCl [25:75 (v/v)] |
| Cleaning/Regeneration: | 1M NaOH |
| Sterlization/Sanitization/Depyrogenation: | 1M NaOH (2 hours); autoclave 30 min 120°C, pH 7.0 |
| Long-term Storage: | 4°C in preservative |
Ordering Information
| Catalog Number | Pack Size |
|---|---|
| 0120-00025 | 25ml |
| 0120-00100 | 100ml |
| 0120-01000 | 1000ml |
For information about larger volumes, please contact us.
MIMETIC BLUE™ AP
Mimetic Blue™ AP is a synthetic affinity adsorbent developed specifically for the purification of alkaline phosphatase from calf intestinal mucosa and other sources.
- ONE STEP PURIFICATION: 300-FOLD INCREASE IN SPECIFIC ACTIVITY
- HIGHLY STABLE LIGAND, RESISTANT TO SODIUM HYDROXIDE
- EASY SCALE-UP
The Mimetic Blue™ AP ligand consists of a blue chromophore linked to a functional phosphonic acid group. This ligand displays a high affinity for calf intestinal alkaline phosphatase and the adsorbent typically provides a one-step purification from crude protein extracts. Increases of specific activity of 300-fold are possible using this media. Mimetic Blue™ AP also provides an excellent separation of phosphodiesterase from alkaline phosphatase.
The affinity ligand is bonded onto 6% cross-linked agarose by a highly stable bonding method which is resistant to treatment with 1M NaOH. The use of NaOH cleaning procedures ensures that Mimetic Blue AP can be used repeatedly for purification of relatively crude protein extracts.
Scale-up is simple. The adsorbent is manufactured to GMP and ISO 9001:2000 standards and large batch sizes are available for process applications. Drug Master Files (DMF) have been submitted for the facility to make traceability and compliance easier.
Specifications
| Support Matrix: | 6% cross-linked agarose (6XL) |
| Bead Size: | 45 - 165 microns |
| Exclusion Limit: | 4 x 106 Daltons (6XL) |
| Temperature Range: | 4 - 120°C |
| pH Range: | 3 - 13 (continuous) 2 - 14 (intermittent) |
| Maximum Pressure: | 1 bar (15 psi) |
| Storage: | 4°C in a solution of ethanol/0.1 M NaCl (25:75 v/v) |
| Cleaning/Regeneration: | 1M NaOH |
| Chemical Compatibility: | Compatible with alcohols, esters, ketones, ethers and most biological buffers; not harmed by common additives such as chaotropes, detergents, chelating agents, thiols |
| Sterlization/Sanitization/Depyrogenation: | 1M NaOH; autoclave 30 min at 120°, pH 7.0 |
| Immobilized Ligand Concentration: | 3 +/- 0.5 micromol/g |
| Alkaline Phosphatase Binding Capacity: | Varies depending on sample purity. Typically 2mg/ml for crude extracts |
Application Examples
1. Purification of alkaline phosphatase from a calf mucosal extract. Extract (17ml in 10mM Tricine buffer pH 8.5) was applied to a 2ml column (1cm ID) of Mimetic Blue™ AP at a flow rate of 0.5ml/min. Bound alkaline phosphatase was eluted with a 5mM phosphate after a stable baseline was reached. A 170-fold increase in specific activity was achieved with 86% of the enzyme activity recovered in the elution peak. Alkaline phosphatase activity was measured at 25°C using p-nitro-phenolphosphate as substrate and 25mM glycine buffer, pH 9.6.
2. Chromatography of commercial "high purity" alkaline phosphatase with a specific activity of 1,200 units/mg. 2ml of a 1mg/ml solution were applied to a 2ml column under the same conditions as described above. The detector response indicates the removal of A280 adsorbing contaminants from the dialyzed high purity enzyme preparation.
Ordering Information
| Catalog Number | Pack Size |
|---|---|
| 0110-00025 | 25ml |
| 0110-00100 | 100ml |
| 0110-01000 | 1000ml |
For information about larger volumes, please contact us.
MIMETIC BLUE™ LIGAND ELISA KIT
For determination of Mimetic Blue™ 1, Reactive Blue 2 and equivalent dye-ligands
- USE AS A QC TEST FOR PRESENCE OF LIGAND IN PURIFIED PROTEIN PRODUCT
- QA/QC FOR INCOMING OR STORED AFFINITY MEDIA
- NANOMOLAR DETECTION LIMITS
ProMetic's enzyme-linked immunosorbent assay (ELISA) for Mimetic Blue™ 1/Reactive Blue 2 is designed for use in monitoring ligand leakage from affinity adsorbents containing Mimetic Blue™ 1, C.I. Reactive Blue 2 or equivalent dye ligands. This assay is an adaptation of the method of Stewart, et. al. and utilizes a purified rabbit polyclonal anti-Mimetic Blue™ 1 antibody in a competitive sandwich ELISA. Detection limits are in the low nanomolar concentration range, several orders of magnitude more sensitive than the limits of spectrophotometric detection.
Description
The ELISA is performed by incubating dilutions of sample and anti-Mimetic Blue™ 1 antibody in PVC 96-well microtiter plates coated with purified Mimetic Blue™ 1-Hemoglobin conjugate. Bound antibody is detected with a goat anti-rabbit IgG peroxidase conjugate in conjunction with a chromogenic assay. Serial dilution of a standard Mimetic Blue™ 1 solution yields a standard curve from which the sensitivity of the assay may be determined and the concentration of Mimetic Blue™ 1 in the samples may be estimated. The effect of interfering protein (e.g. eluted product) on the apparent free ligand concentration can be determined by constructing a standard curve in the presence of potentially competing protein. Any difference between spiked and un-spiked standard curves is due to dye sequestration by the protein(s).
This assay has a detection limit of approximately 10nMolar (1 x 1011 moles per ml; 10 ppb).
Because certain similarities exist between the structures of Mimetic Blue™ 1 and the reactive textile dye C.I. Reactive Blue 2, this assay may also be used to detect leakage from affinity supports containing C.I. Reactive Blue 2 (isomers-or isomeric mixtures which are also known by the trade names Procion Blue H-B, Cibacron Blue F-3GA and Cibacron Blue 3GA). It is strongly recommended that separate standard curves are prepared for each dye ligand tested because of the variations in these dyes from various sources (an example of a standard curve is shown below).
Reference
Reprinted from J. Chrom., 623, Stewart et. al., “Development of an enzyme-linked immunosorbent assay for C.I. Reactive Blue 2 and its application to a comparison of the stability and performance of perfluorocarbon support with other immobilized C.I. Reactive Blue 2 affinity adsorbents”. 1-14, 1992., with kind permission from Elsevier Science – NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.
Ordering Information
Catalog Number: 2090
10 plate assays (20 sample assays)
The ProMetic ELISA/Blue Kit contains 2mg of Mimetic Blue™ 1-Hemoglobin conjugate, 0.25ml of Rabbit anti-Mimetic Blue l, 0.2ml of Anti-rabbit IgG-peroxidase conjugate (Sigma A-6154) and 1ml of standard 100µM Mimetic Blue™ 1 solution (supplied as 2 x 0.5ml) to perform 10 x 96-well microtiter plate assays.
Please note that additional standard laboratory reagents are required to perform the assay but are not included in the ELISA kit.
For more information, please contact us.