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FLAG® Western Detection Kit
INSTRUCTION MANUAL
Catalog #200470
Revision A
For In Vitro Use Only
200470-12
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LIMITED PRODUCT WARRANTY
This warranty limits our liability to replacement of this product. No other warranties of any kind,
express or implied, including without limitation, implied warranties of merchantability or fitness for
a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect,
consequential, or incidental damages arising out of the use, the results of use, or the inability to use
this product.
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FLAG® Western Detection Kit
CONTENTS
Materials Provided.............................................................................................................................. 1
Storage Conditions.............................................................................................................................. 1
Additional Materials Required .......................................................................................................... 2
Western Blotting and Antibody Probing of Electroblots....................................................... 2
Color Development Reaction ................................................................................................ 2
Dot Blot Assay ...................................................................................................................... 2
Notices to Purchaser ........................................................................................................................... 3
Introduction......................................................................................................................................... 4
Western Blotting and Antibody Probing of Electroblots................................................................. 4
Color Development Reaction ............................................................................................................. 6
Preparation of the Color Development Solution Containing Nitroblue Tetrazolium (NBT)
and 5-Bromo-4-chloro-3-indolyl Phosphate (BCIP) .....................................................6
Color Development on the Nitrocellulose Membrane........................................................... 6
Dot Blot Assay (Optional)................................................................................................................... 7
Troubleshooting .................................................................................................................................. 9
Western Blotting, Antibody Probing of Electroblots, and Color Development Reaction..... 9
Dot Blot Assay (Optional)..................................................................................................... 9
Preparation of Reagents ................................................................................................................... 10
Stock Solutions.................................................................................................................... 10
Standard Reagents ...............................................................................................................10
References .......................................................................................................................................... 11
Endnotes............................................................................................................................................. 11
MSDS Information............................................................................................................................ 11
Quick-Reference Protocol ................................................................................................................ 14
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FLAG® Western Detection Kit
ATERIALS PROVIDED
M
Materials provideda Quantity
Anti-FLAG M2 antibody
Goat anti-mouse antibody, AP conjugated, immunoaffinity purified, polyclonalc 100 μl
Sterile, filtered fraction V bovine serum albumin (BSA) [30% (w/v) in saline] 15 ml
Nitroblue tetrazolium (NBT)b [75 mg/ml in 70% N,N-dimethylformamide (DMF)] 2 ml
5-Bromo-4-chloro-3-indolyl phosphate (BCIP)b (50 mg/ml in 100% N,N-DMF) 1.5 ml
a
Sufficient materials are provided for ten 100-mm nitrocellulose membranes.
b
Also available separately [Anti-FLAG M2 antibody, Stratagene Catalog #200471 (200 μg), #200472 (1 mg);
BCIP (5-bromo-4-chloro-3-indolyl phosphate, Stratagene Catalog #300045 (1.5 ml);
NBT (nitroblue tetrazolium), Stratagene Catalog #300130 (2 ml)].
c
Please see the Certificate of Analysis for the concentration.
d
Immediately on receipt, store the anti-FLAG M2 antibody at –20°C and the goat anti-mouse antibody at 4°C. Avoid
multiple freeze–thaw cycles. Aliquot and fast freeze the primary and secondary antibodies for long-term storage.
b–d
200 μg
STORAGE CONDITIONS
BSA: –20°C
Anti-FLAG
®
M2 Antibody: –20°C
Note For best results, store the anti-FLAG M2 antibody at –20°C.
Goat Anti-Mouse Antibody: 4°C
NBT: 4°C
BCIP: 4°C
Revision A © Agilent Technologies, Inc. 2009.
FLAG® Western Detection Kit
1
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ADDITIONAL MATERIALS REQUIRED
Western Blotting and Antibody Probing of Electroblots
Laemmli sample buffer (1×)§
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) system
Nitrocellulose membranes (available from Stratagene, Catalog #420106, #420107, and #420108)
Transfer buffer (1×)§
Blocking solution
Tris-buffered saline (TBS)§
Color Development Reaction
Notes Do not prepare the color development solution containing the NBT and BCIP until
Color development solution
Tris-buffered saline (TBS)
Stop solution
®
Whatman
3MM paper
Dot Blot Assay
Strip of nitrocellulose membrane (1 × 6 cm)
Blocking solution or
Tris-buffered saline (TBS)
Color development solution
®
Whatman
3MM paper
Stop solution
§
See Preparation of Reagents.
§
or bovine lacto transfer technique optimizer (BLOTTO)
§
just prior to use in the color development reaction [see Preparation of the Color
Development Solution Containing Nitroblue Tetrazolium (NBT) and 5-Bromo4-chloro-3-indolyl Phosphate (BCIP) subsection of Color Development Reaction].
§
§
BLOTTO
2
FLAG® Western Detection Kit