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Kits  >  Antibodies to FLAG™ and Antibodies to 6XHIS Tags
 

TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag

Monoclonal
Code

88-8887-383
Size

1 Kit
Price

$399.00
Availability

Ships next business day.
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Phone: 800.656.7625
E-mail: orders@rockland-inc.com
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Immunoprecipitation of VHH-EGFR-FLAG single chain antibody.
500 ug of Hela Whole Cell Lysate (p/n W09-000-364) spiked with 1 ug of VHH-EGFR-FLAG single chain antibody was immunoprecipitated with 1 ug of Mouse anti-FLAG® Monoclonal Antibody (p/n 200-301-383) overnight at 4°C. Western Blotting of Immunoprecipitation product followed. Lane 1 - VHH-EGFR-FLAG Immunoprecipitation. Lane 2 - VHH-EGFR-FLAG Pre-Cleared Lysate (prior to immunoprecipitation). Load: 2 uL per lane. Primary Antibody: Mouse anti-FLAG® Monoclonal Antibody at 1:1,000 o/n at 4°C. Secondary Antibody: Mouse Trueblot® ULTRA: Anti-Mouse Ig HRP at 1:2,000 for 1 HR at RT. Block: MB-070 for 2 HR at RT. Predicted/Observed size: 11 kDa, 11 kDa for VHH-EGFR-FLAG single chain antibody. Enrichment is evident after immunoprecipitation (Lane 1) as opposed to starting material (Lane 2).
Monoclonal Antibody to detect FLAG™ conjugated proteins detects both C terminal linked and N terminal linked FLAG™ tagged recombinant proteins by western blot.
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Shipping:
$85.00 to United States
Handling:
$20.00
Synonyms: HRP, TrueBlot, HRP TrueBlot ULTRA, Peroxidase TrueBlot, TrueBlot for IP/WB, TrueBlot for immunoprecipitation, TrueBlot for western blotting, Flag Western Blot, Western Blotting Kit, Chemiluminescent Flag Kit, Immunoblotting Kit, FLAG®, HRP Anti FLAG

TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag Properties

Description
TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag - 88-8887-383
Target Species
Mouse
Known Cross Reactivity
FLAG®, Mouse
Clonality
Monoclonal
Application
Western Blot : 1:1000
ImmunoPrecipitation: 1-10 µg / 10^7 cells/1 mL lysate
Product Type
Kit
Kit Type
Immunoprecipitation Kit
Physical State
Liquid (sterile filtered)
Shipping condition
Wet Ice
Format
IgG
Buffer
0.01 M Sodium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Preservative
Wash buffers MUST NOT contain SODIUM AZIDE or other inhibitors of peroxidase activity!
Stabilizer
0.1 mg/ml Bovine Serum Albumin (BSA) - IgG and Protease free, 50% (v/v) Glycerol
Related Links
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TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag Description

Background
The FLAG® epitope is a small but highly immunogenic peptide DYKDDDDK (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) which allows fusion proteins to retain their original conformation and function. The hydrophilic character of this sequence increases the likelihood that it will be located on the surface of FLAG® -tagged recombinant proteins for reaction with anti-DYKDDDDK (FLAG®) antibodies. The anti-DYKDDDDK (FLAG®) antibody included in this Western blot kit detects FLAG® -tagged recombinant proteins fused with FLAG® tag either at the amino-terminus or carboxy-terminus of the protein. The TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag contains the critical supporting reagents, buffers, and substrates for immunoprecipitation and Western blotting of samples containing the DYKDDDDK (FLAG®) Epitope Tag using TrueBlot second step immunoblotting reagents in conjunction with Rockland's Antibody for the detection of FLAG™ conjugated proteins (MOUSE) Monoclonal Antibody. TrueBlot technology enables unhindered detection of protein bands of interest which would otherwise be obscured by the presence of reduced and denatured heavy and light chain immunoglobulin in the blot (as detected by the conventional immunoblotting HRP anti-mouse IgG reagent). Mouse IgG TrueBlot® ULTRA is the unique horseradish peroxidase conjugated anti-mouse IgG immunoblotting second step reagent which enables detection of immunoblotted target protein bands, without hindrance by interfering immunoglobulin heavy and light chains from your IP antibody. Use it in place of your usual HRP anti-mouse IgG immunoblotting second step reagent. It is easy to generate publication-quality IP/WB data with Mouse IgG TrueBlot® ULTRA. Mouse IgG TrueBlot ULTRA is ideal for use in protocols involving immunoblotting of immunoprecipitated proteins. TrueBlot preferentially detects the non-reduced form of mouse IgG over the reduced, SDS-denatured form of IgG. When the immunoprecipitate is fully reduced immediately prior to SDS-gel electrophoresis, reactivity of Mouse IgG TrueBlot ULTRA with the 55 kDa heavy chains and the 23 kDa light chains of the immunoprecipitating antibody is minimized thereby eliminating interference by the heavy and light chains of the immunoprecipitating antibody in IP/immunoblotting applications. Applications include studies examining post-translational modification (e.g., phosphorylation or acetylation) or protein-protein interactions.
Storage Condition
Store at 2-8°C, except Mouse TrueBlot® ULTRA and Antibody for the detection of FLAG™ conjugated proteins (MOUSE) Monoclonal Antibody, which should be stored at -20°C. This product is guaranteed for 6 months upon receipt, when handled and stored as instructed.
Application Note
Mouse IgG TrueBlot® ULTRA is provided as 1000X solution. In order to conserve reagent, we recommend incubating the blots from minigels in sealed bags (removing as much air as possible) with minimal volume (2-3 mls). Mouse IgG TrueBlot® ULTRA. An HRP-conjugated second step reagent reacting with mouse IgGs for optimal signal detection in immunoprecipitation/immunoblotting experiments. Note that there are three key procedural considerations: 1. Protein A or G beads may be used with the mouse, goat and sheep TrueBlot secondaries but not with the rabbit TrueBlot secondary. Use of protein A or G beads with the rabbit TrueBlot will result in contaminating bands. 2. Immunoprecipitate should be completely reduced. 3. Milk should be used as the blocking protein for the immunoblot. The TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag components are sufficient for 20-25 miniblots. Components: 1. Mouse IgG TrueBlot ULTRA: 50 µL 18-8817-31 2. TrueBlot Enhancer Solution: 25 mL 3. TrueBlot Blocker: 10 g 4. TrueBlot Assay Buffer: 30 mL 20X 5. TrueBlot Substrate A: 12.5 mL 6. TrueBlot Substrate B: 12.5 mL 7. Anti-Mouse Ig IP Beads: 2.5 mL Binds 0.4 mg Ig/mL beads 8. Antibody for the detection of FLAG™ conjugated proteins (MOUSE) Monoclonal Antibody: 100 µg 9. Western Blot Incubation Tray Special Notes: Upon initial use of the IP beads, we recommend that the vial be inverted several times to get the beads into suspension. We recommend to use a large bore pipet to pipet up the liquid for use. For storage of the opened vial of beads, we recommend that the vial cap be sealed with parafilm to help prevent evaporation of the buffer.
Purity/Specifity
This TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag allows for the detection of FLAG® -tagged recombinant protein present in cell lysates provided by the user. Mouse TrueBlot® ULTRA Antibody Peroxidase Conjugate was prepared from tissue culture supernatant by Protein G affinity chromatography. Assay by Immunoelectrophoresis resulted in a single precipitin arc against anti-Mouse Serum. Reactivity is observed against native Mouse IgG by both Western blot and ELISA.
Disclaimer Note-General
This product is for research use only and is not intended for therapeutic or diagnostic applications. Please contact a technical service representative for more information. All products of animal origin manufactured by Rockland Immunochemicals are derived from starting materials of North American origin. Collection was performed in United States Department of Agriculture (USDA) inspected facilities and all materials have been inspected and certified to be free of disease and suitable for exportation. All properties listed are typical characteristics and are not specifications. All suggestions and data are offered in good faith but without guarantee as conditions and methods of use of our products are beyond our control. All claims must be made within 30 days following the date of delivery. The prospective user must determine the suitability of our materials before adopting them on a commercial scale. Suggested uses of our products are not recommendations to use our products in violation of any patent or as a license under any patent of Rockland Immunochemicals, Inc. If you require a commercial license to use this material and do not have one, then return this material, unopened to: Rockland Inc., P.O. BOX 326, Gilbertsville, Pennsylvania, USA.
General Reference
Kong, D., L. Xu, Y. Yu, W. Zhu, D.W. Andrews, Y. Yoon, and T.H. Kuo. 2005. Regulation of Ca2+-induced permeability transition by BCL-2 is antagonized by Drp1 and hFis1. Molecular and Cellular Biochemistry. 272: 187-199. (Rabbit IgG TrueBlot, PubMed) DiPerna, G., J. Stack, A.G. Bowie, A. Boyd, G. Kotwal, Z. Zhang, S. Arvikar, E. Latz, K.A. Fitzgerald, and W.L. Marshall. 2004. Poxvirus protein N1L targets the I-kappaB Kinase complex, inhibits signaling to NF-kappaB by the Tumor Necrosis Factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by Toll-like Receptors. J. Biol. Chem. 279: 36570-36578. (Rabbit IgG TrueBlot, PubMed) Zhang, X., Y. Ozawa, H. Lee, Y. Wen, T. Tan, B. Wadzinski, and E. Seto. 2005. Histone deacetylase 3 (HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4. Genes & Development. 19: 827-839. (Rabbit IgG TrueBlot, PubMed) Lehtonen, S., E. Lehtonen, K. Kudlicka, H. Holthöfer, and M.G. Farquhar. 2004. Nephrin Forms a Complex with Adherens Junction Proteins and CASK in Podocytes and in Madin-Darby Canine Kidney Cells Expressing Nephrin. Am J Pathol. 165:923-936. (Rabbit IgG TrueBlot, PubMed) Tyagi A, Agarwal C, Harrison G, Glode LM, Agarwal R. 2004. Silibinin causes cell cycle arrest and apoptosis in human bladder transitional cell carcinoma cells by regulating CDKI-CDK-cyclin cascade, and caspase 3 and PARP cleavages. Carcinogenesis. 25: 1711-20. (Mouse IgG TrueBlot, PubMed) Antibodies, A Laboratory Manuel. Ed Harlow and David Lane, eds. Cold Spring Harbor Press. 1988. Chapter 12 gives an excellent overview of Western Blotting techniques, including India Ink staining. Current protocols in Molecular Biology. J. Ausebel, et al, eds. John Wiley and Sons, New York. Gives a complete protocol of Western Blotting and Dot Blotting. Molecular Cloning: A Laboratory Manuel. 2nd Edition. J. Sambrook, E.F. Fritsch and T. Maniatis, eds. Cold Spring Harbor Press, 1989. Chapter 18 gives detailed protocols for both the production of cell lysates and electrophoresis and blotting of proteins
Specific Reference
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Product Type
GFP and Epitopes;
Applications
ELISA, WB, IHC, FC
Reacts With
Carboxy and amino terminal linked FLAG™ tagged recombinant proteins
Catalog Number
200-301-383

Product Type
Proteins and Peptides;
Applications
WB, IP
Catalog Number
000-000-383

Product Type
Secondary Antibodies;
Applications
WB
Reacts With
Mouse
Catalog Number
18-8817-30

Product Type
Secondary Antibodies;
Applications
WB
Reacts With
Mouse
Catalog Number
18-8817-31

Product Label

 

Description
HRP TrueBlot® ULTRA for IP/WB
Color
Conjugation Reference
Molecular Weight
Excitation Wavelength
Conjugation Chemistry