Revision 14

#97135Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IHC-P, IF-F, FC-FP, ChIP, C&R

REACTIVITY:

H M

SENSITIVITY:

Endogenous

MW (kDa):

62

Source/Isotype:

Rabbit IgG

UniProt ID:

#Q9JKD8

Entrez-Gene Id:

57765

Product Information

Product Usage Information

For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:200
Immunohistochemistry (Paraffin) 1:100 - 1:400
Immunofluorescence (Frozen) 1:400 - 1:1600
Flow Cytometry (Fixed/Permeabilized) 1:100 - 1:400
Chromatin IP 1:50
CUT&RUN 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #53753.

Specificity / Sensitivity

T-bet/TBX21 (E4I2K) Rabbit mAb recognizes endogenous levels of total T-bet/TBX21 protein. E412K non-specifically labels the submucosa and external muscularis of mouse colon by immunofluorescence.

Species Reactivity:

Human, Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a recombinant protein fragment derived from mouse T-bet/TBX21 protein.

Background

The T-box gene family consists of transcription factors characterized by a related DNA-binding domain (T-box) of approximately 200 amino acids (1,2). The T-box genes exhibit diverse temporal and spatial patterns in the developing embryo. Studies have demonstrated members of this family play crucial roles during embryogenesis in a wide range of organisms by regulating cell fate decisions to establish the early body plan and to regulate later processes underlying organogenesis (3-5). Mutations in T-box genes are associated with many developmental defects (6). Recent studies also indicate potential roles in cancer by members of the T-box family (7-9).

  1. Wilkinson, D.G. et al. (1990) Nature 343, 657-9.
  2. Papaioannou, V.E. and Silver, L.M. (1998) Bioessays 20, 9-19.
  3. Showell, C. et al. (2004) Dev Dyn 229, 201-18.
  4. Papaioannou, V.E. (2001) Int Rev Cytol 207, 1-70.
  5. Hoogaars, W.M. et al. (2007) Cell Mol Life Sci 64, 646-60.
  6. Baldini, A. (2004) Curr Opin Cardiol 19, 201-4.
  7. Abrahams, A. et al. (2010) IUBMB Life 62, 92-102.
  8. Rowley, M. et al. (2004) J Mammary Gland Biol Neoplasia 9, 109-18.
  9. Yang, X.R. et al. (2009) Nat Genet 41, 1176-8.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-F: Immunofluorescence (Frozen) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 14
#97135

T-bet/TBX21 (E4I2K) Rabbit mAb

Western Blotting Image 1: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Western blot analysis of extracts from various cell lines and mouse primary CD4+ T cells using T-bet/TBX21 (E4I2K) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, T-bet/TBX21 protein is not detected in EL4 cells.
Western Blotting Image 2: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human T-bet/TBX21 protein (hT-bet/TBX21-Myc/DDK; +) and Myc/DDK-tagged full-length mouse T-Bet/TBX21 protein (mT-bet/TBX21-Myc/DDK; +), using T-bet/TBX21 (E4I2K) Rabbit mAb (upper), DYKDDDDK Tag Antibody #2368 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunoprecipitation Image 1: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunoprecipitation of T-bet/TBX21 protein from NK-92 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is T-bet/TBX21 (E4I2K) Rabbit mAb. Western blot analysis was performed using T-bet/TBX21 (E4I2K) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.
Immunohistochemistry Image 1: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded 293T cell pellets, control (left) or mouse T-bet/TBX21-transfected (right), using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 2: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded normal human spleen using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 3: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 4: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human small cell carcinoma of the salivary gland using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 5: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human gastric carcinoma using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 6: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse thymus using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 7: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse lung using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 8: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse ovary using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 9: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded CT26.WT syngeneic tumor using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 10: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded LL/2 syngeneic tumor using T-bet/TBX21 (E4I2K) Rabbit mAb.
Immunohistochemistry Image 11: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using T-bet/TBX21 (E4I2K) Rabbit mAb (left) or T-bet/TBX21 (D6N8B) XP® Rabbit mAb (right) #13232. These two antibodies detect unique, non-overlapping epitopes on human T-bet/TBX21. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemistry Image 12: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human B-cell non-Hodgkin lymphoma using T-bet/TBX21 (E4I2K) Rabbit mAb (left) or T-bet/TBX21 (D6N8B) XP® Rabbit mAb (right) #13232. These two antibodies detect unique, non-overlapping epitopes on human T-bet/TBX21. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemistry Image 13: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse spleen using T-bet/TBX21 (E4I2K) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).
Immunofluorescence Image 1: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of mouse thymus using T-bet/TBX21 (E4I2K) Rabbit mAb (red), CD8α (2.43) Rat mAb (FITC Conjugate) #35467 (green), and CD45 (D3F8Q) Rabbit mAb (Alexa Fluor® 647 Conjugate) #81143 (blue pseudocolor).
Flow Cytometry Image 1: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Flow cytometric analysis of K-562 cells (blue, negative) and NK-92 (green, positive) using T-bet/TBX21 (E4I2K) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow Cytometry Image 2: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Flow cytometric analysis of EL4 cells (blue, negative) and CTLL-2 cells (green, positive) using T-bet/TBX21 (E4I2K) Rabbit mAb (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from NK-92 cells and either T-bet/TBX21 (E4I2K) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human CCL4 promoter primers, SimpleChIP® Human IFN-γ Promoter Primers #13051, 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.
CUT and RUN Image 1: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
CUT&RUN was performed with NK-92 cells and T-bet/TBX21 (E4I2K) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across CKS1B, a known target gene of T-bet (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
CUT&RUN was performed with NK-92 cells and T-bet/TBX21 (E4I2K) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 1 (upper), including CKS1B (lower), a known target gene of T-bet (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: T-bet/TBX21 (E4I2K) Rabbit mAb Expand Image
CUT&RUN was performed with NK-92 cells and either T-bet/TBX21 (E4I2K) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human CKS1B exon 1 primers and human KC6 upstream primers. 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.