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To Purchase # 9962T
| 9962T | 1 Kit (5 x 20 µl) | $359.00 |
| $0.00 | ||
Epitope Tag Antibody Sampler Kit #9962
Western blot analysis of extracts from COS-7 cells untransfected (-) and transfected cells overexpressing GST-Bad (+), using GST (91G1) Rabbit mAb (left) and Bad Antibody #9292 (right).
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Western blot analysis of extracts from untransfected control cells (-) and transfected cells overexpressing Myc-Bcl-2 (+), using Bcl-2 Antibody #2872 (left) and Myc-Tag (71D10) Rabbit mAb (right).
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Western blot analysis of extracts from HeLa cells, untransfected or transfected with either HA-FoxO4 or HA-Akt3, using HA-Tag (C29F4) Rabbit mAb.
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Western blot analysis of extracts from 293T cells, untransfected or transfected with 6xHis-Tag fusion protein 1 or 6xHis-Tag fusion protein 2, using His-Tag (D3I1O) XP® Rabbit mAb.
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Western blot analysis of extracts from 293T cells, untransfected or transfected with either DYKDDDDK-tagged FoxG1, NPAS4, or Tyrosine Hydroxylase (TH), using DYKDDDDK Tag (9A3) Mouse mAb.
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After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
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Flow cytometric analysis of COS cells, untreated (blue) or Myc transfected (green), using Myc-Tag (71D10) Rabbit mAb.
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Immunohistochemical analysis of paraffin-embedded COS cells, untransfected (right) or HA-Tag transfected (left), using HA-Tag (C29F4) Rabbit mAb.
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Immunoprecipitation of His-Tag protein from transfected 293T cells using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or His-Tag (D3I1O) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using His-Tag (D3I1O) XP® Rabbit mAb.
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Immunohistochemical analysis of paraffin-embedded 293T cell pellets, control (left) or ROS-DYKDDDDK Tag transfected (right), using DYKDDDDK Tag (9A3) Mouse mAb.
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Immunocytochemical staining of cells expressing Myc-Bcl-2 protein (left) or cells without transfected protein (right), using Myc-tag (71D10) Rabbit mAb.
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Flow cytometric analysis of HeLa cells, untransfected (blue) or transfected with HA-FoxO4 (green), using HA-Tag (C29F4) Rabbit mAb #3724.
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Flow cytometric analysis of 293T cells, untransfected (blue) or transfected with a His-myc-Akt plasmid (green), using His-Tag (D3I1O) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.
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Flow cytometric analysis of Cos cells, untreated (blue) or transfected with a DYKDDDDK-tagged IRF3 contruct (green) using DYKDDDDK Tag (9A3) Mouse mAb.
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Confocal immunofluorescent analysis of COS cells, transfected with an HA-tagged protein (left) or mock-transfected (right), using HA-Tag (C29F4) Rabbit mAb (green). Actin filaments have been labeled using DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® (fluorescent DNA dye).
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Confocal immunofluorescent analysis of 293T cells transfected with a His-Tagged protein using His-Tag (D3I1O) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).
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Confocal immunofluorescent analysis of HeLa cells transfected with a DYKDDDDK-tagged IRF3 construct using DYKDDDDK Tag (9A3) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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293T cells were either untransfected (left panel) or transfected with an HA-tagged human CBP construct (right panel), then treated with Forskolin #3828 (30 µM) . Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 cells and 10 µl of HA-Tag (C29F4) Rabbit mAb, 10 µl of CBP (D9B6) Rabbit mAb, or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human ALS2 exon 1 primers, SimpleChIP® Human NR4A3 Promoter Primers #4829, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
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293T cells were either untransfected (left panel) or transfected with an His-tagged human Stat3 construct (right panel), then treated with Human Interleukin-6 (hIL-6) #8904 (100 ng/ml, 30 min). Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 cells and 5 µl of His-Tag (D3I1O) XP® Rabbit mAb, 5 µl of Stat3α (D1A5) XP® Rabbit mAb #8768, or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human c-Fos Promoter Primers #4663, human IRF-1 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
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| Product Includes | Quantity | Applications | Reactivity | MW(kDa) | Isotype |
|---|---|---|---|---|---|
| GST (91G1) Rabbit mAb 2625 | 20 µl |
|
All | Rabbit IgG | |
| Myc-Tag (71D10) Rabbit mAb 2278 | 20 µl |
|
Rabbit IgG | ||
| HA-Tag (C29F4) Rabbit mAb 3724 | 20 µl |
|
All | Rabbit IgG | |
| His-Tag (D3I1O) XP® Rabbit mAb 12698 | 20 µl |
|
All | Rabbit IgG | |
| DYKDDDDK Tag (9A3) Mouse mAb (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) 8146 | 20 µl |
|
Mouse IgG1 | ||
| Anti-rabbit IgG, HRP-linked Antibody 7074 | 100 µl |
|
Goat | ||
| Anti-mouse IgG, HRP-linked Antibody 7076 | 100 µl |
|
Horse |
Product Usage Information
Protocols
2278: Western Blotting, Immunoprecipitation (Magnetic), Immunofluorescence, Flow
2625: Western Blotting, Immunoprecipitation (Magnetic)
3724: Western Blotting, Immunoprecipitation (Magnetic), Immunohistochemistry (Paraffin), Immunofluorescence, Flow, ChIP Magnetic
7074: Western Blotting
7076: Western Blotting
8146: Western Blotting, Immunoprecipitation (Magnetic), Immunohistochemistry (Paraffin), Immunofluorescence, Flow
12698: Western Blotting, Immunoprecipitation (Magnetic), Immunofluorescence, Flow, ChIP Magnetic
Product Description
The Epitope Tag Antibody Sampler Kit provides an economical means to analyze the expression of a variety of epitope tagged proteins. The kit contains enough primary and secondary antibodies to perform four Western blots per primary antibody.
Specificity / Sensitivity
All antibodies in the Epitope Tag Antibody Sampler Kit detect overexpressed fusion proteins containing the corresponding epitope tags. DYKDDDDK Tag Antibody recognizes the DYKDDDDK peptide (the same epitope recognized by Sigma’s Anti-FLAG® antibodies), and its binding specificity is NOT dependent on the presence of divalent metal cations.
Source / Purification
Rabbit monoclonal antibodies are producted by immunizing rabbits with a GST fusion protein, a synthetic peptide corresponding to residues 410-419 of human c-Myc (EQKLISEEDL), residues of the 6xHis epitope tag, or with a synthetic peptide containing the influenza hemagglutinin epitope (YPYDVPDYA). Mouse monoclonal antibodies are produced by immunizing animals with a synthetic DYKDDDDK peptide. Polyclonal antibodies are produced by immunizing rabbits with a synthetic DYKDDDDK peptide. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Background
Epitope tags are useful for the labeling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Because of their small size, they are unlikely to affect the tagged protein’s biochemical properties.
Several different epitope tags are now commonly utilized and readily available. For instance, a variety of plasmids contain DNA that encodes an amino-terminal tag consisting of six histidine (6xHis) residues followed by an extended multiple cloning site. The 6xHis tag on the expressed recombinant proteins allows for efficient coupling to Ni2+ affinity resins and purification by single step chromatography (1). As is the case with other protein tag systems (2), this polyhistidine tag can often be cleaved at sites recognized by proteases such as thrombin and enterokinases to isolate the protein of interest (1). Glutathione S-transferase (GST) is another widely used fusion partner, since it provides both an easily detectable Tag and a simple purification process with little effect on the biological function of the protein of interest. Numerous vectors containing GST-Tag have been developed for both prokaryotic and eukaryotic systems over the past decade (3-5). The HA tag, derived from an epitope of the influenza hemagglutinin protein, has also been extensively used as a general epitope tag in expression vectors (6), while the Myc epitope tag is routinely used to detect expression of recombinant proteins in bacteria, yeast, insect and mammalian cell systems (7). Finally, the DYKDDDDK peptide has been used extensively as a general epitope tag in expression vectors and consists of only eight amino acids. This peptide can be expressed and detected with the protein of interested as an amino-terminal or carboxy-terminal fusion (8).
1. Kroll, D.J. et al. (1993) DNA Cell Biol. 12, 441-453.
2. Brizzard, B. L. et al. (1994) Biotechniques 16, 730-735.
3. di Guan, C. et al. (1988) Gene 67, 21-30.
4. Guan, K.L. and Dixon, J.E. (1991) Anal. Biochem. 192, 262-267.
5. Davis, A.H. et al. (1993) Biotechnology 11, 933-936.
6. Yu, J. et al. (1998) Mol. Cell. Biol. 18, 1379-1387.
Data Sheets & Documentation
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 5,675,063.
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