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ANTI-FLAG® M2 Affinity Gel
A2220
Storage Temperature −20 °C
Product Description
ANTI-FLAG® M2 Affinity Gel is a purified murine IgG1
monoclonal antibody covalently attached to agarose
by a hydrazide linkage. It is useful for purification or
immunoprecipitation of FLAG® fusion proteins.
ANTI-FLAG® M2 binding to the FLAG® peptide is not
dependent on calcium.
Binding Specificity:
FLAG® octapeptide
(N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) at
N-terminal, Met-N-terminal, C-terminal, and
internal locations of a fusion protein.
Reagent
ANTI-FLAG® M2 Affinity Gel is supplied as a
50% suspension in 50% glycerol with 10 mM sodium
phosphate and 150 mM sodium chloride, pH 7.4,
containing 0.02% (w/v) sodium azide (PBS/A).
Equipment and Reagents
(Required but not provided)
• Cells expressing FLAG® fusion protein
• Lysis buffer (50 mM Trizma®-HCl, pH 7.4, with
150 mM NaCl, 1 mM EDTA, and 1% TRITON®
X-100), CelLytic M (Cat. No. C2978), or
CelLytic B (Cat. No. B7435, B7310, or C8740)
• Appropriate centrifuge
• Appropriate column or centrifuge tubes
• Sodium chloride (e.g. Cat. No. S3014)
• Trizma base (e.g. Cat. No. T6066)
• Protease inhibitor cocktail for use with
mammalian cells and tissue extracts
(Cat. No. P8340)
Storage/Stability
ANTI-FLAG® M2 Affinity Gel should be stored in
50% glycerol at –20 °C for maximum stability. The
unopened product is stable for one year when stored
as indicated. After use, the resin should be cleaned
and stored in 50% glycerol with TBS or PBS buffer
containing 0.02% sodium azide to protect the
product. Do not freeze in the absence of glycerol.
Procedure
Note: It is recommended that the entire Technical
Bulletin be read before use, especially the Reagent
Compatibility Table.
Part I. Cell Lysate Preparation
The researcher must empirically determine the most
suitable procedure. Typical methods for purifying
FLAG® fusion proteins from crude E. coli extracts are
provided. It is recommended that the CelLytic B
Lysis Reagents (Cat. No. B7435, B7310, or C8740) or
CelLytic B Plus Kit (Cat. No. CB0050 or CB0500)
products be used for bacterial cell lysis. CelLytic M
can be used for mammalian cells.
Recommended procedure for E. coli using
CelLytic Lysis Reagents
1. Grow the cells ( 1 liter or less) under conditions
that induce production of FLAG® fusion proteins.
2. Harvest the cells by centrifugation at 5,000 g for
30 minutes at 2−8 °C.
3. Decant the medium from the cell paste.
4. Freeze the cell paste using a dry ice/ethanol bath
or at –20 °C in a freezer. Cell lysis is enhanced
during the slow freezing.
5. Lyse the frozen cells with 10 mL of CelLytic B
(Cat. No. B7435) per g of frozen cell paste or
5 mL of CelLytic B, 2 concentrate
(Cat. No. B7310) per g of frozen cell paste.
6. Resuspend the cells in the CelLytic B reagent
with a pipette. Mix vigorously on a stir plate for
15 minutes to fully extract the protein. |