Exbio — Research products — Antibodies — Other intracellular antigens — Anti-SIT Purified

Anti-SIT Purified

Regulatory status

RUO

Antigen

SIT

Clone

SIT-01

Format

Purified

Reactivity

Human

Application

FC(IC), IP, WB

Variant

0.1 mg

11-345-C100

In stock

176.00 USD

0.025 mg

11-345-C025

Delivery 1 week

88.00 USD

Variant

0.1 mg

11-345-C100

In stock

176.00 USD

0.025 mg

11-345-C025

Delivery 1 week

88.00 USD

Contact distributor

Product details

Description

Images

References

SDS download

Isotype

Mouse IgG1

Specificity

The antibody SIT-01 reacts with an intracellular epitope of SHP2-interacting transmembrane adaptor protein (SIT) expressed exclusively in lymphoid organs. It weakly crossreacts with murine SIT.

Application

FC(IC), IP, WB

Application details

Flow cytometry: Recommended dilution: 1-5 μg/ml, intracellular staining.
Western blotting: Recommended dilution: 1-2 μg/ml, reducing conditions.

Reactivity

Human

Immunogen

Bacterially produced recombinant intracellular fragment of human SIT.

Concentration

1 mg/ml

Preparation

Purified by protein-A affinity chromatography.

Formulation

Phosphate buffered saline (PBS), pH 7.4, 15 mM sodium azide

Storage and handling

Store at 2-8°C. Do not freeze.

The product is intended For Research Use Only. Diagnostic or therapeutic applications are strictly forbidden. Products shall not be used for resale or transfer to third parties either as a stand-alone product or as a manufacture component of another product without written consent of EXBIO Praha, a.s. EXBIO Praha, a.s. will not be held responsible for patent infringement or any other violations of intellectual property rights that may occur with the use of the products. Orders for all products are accepted subject to the Term and Conditions available at www.exbio.cz. EXBIO, EXBIO Logo, and all other trademarks are property of EXBIO Praha, a.s.

Other names

SIT1

Antigen description

SIT is a 21 kDa transmembrane adaptor protein expressed exclusively in lymphoid organs. It acts either as a positive or as a negative regulatory element in T cell activation and in T cell development. Binding to Grb2 plays a pivotal role in signal transduction. Hubener et al. (2001) determined that the SIT gene contains 5 exons and spans 1.8 kb of genomic DNA. The SIT promoter demonstrated strong transcriptional activity and potential binding sites for both ubiquitous and lymphoid-specific transcription factors.

Entrez Gene ID 27240

UniProt ID Q9Y3P8

Anti-SIT Purified (clone SIT-01) works in WB application.

Western blotting analysis was performed on whole cell extracts (RIPA lysis buffer) of Ramos and Jurkat cell lines, mixed and heated (100°C, 5 min) with reducing (2-mercaptoethanol) SDS-loading buffer. Samples were resolved using 10% SDS-PAGE gel.

Nitrocellulose membrane blot was probed simultaneously with mouse IgG1 monoclonal antibody SIT-01 (2 µg/ml), and rat IgG2a anti-tubulin monoclonal antibody YOL1/34 (1 µg/ml) used as the loading control. Subclass-specific secondary antibodies IRDye 800CW Goat-anti-Rat IgG (green) and IRDye 680LT Goat-anti-Mouse IgG (red) were used for multiplex fluorescent Western blot detection.

SIT was detected at ~32 kDa in tested cell lines.

Flow cytometry multicolor intracellular staining of human peripheral whole blood stained using anti-SIT (SIT-01) purified antibody (concentration in sample 9 μg/ml, GAM APC) and anti-human CD3 (UCHT1) Pacific Blue™ antibody (20 μl reagent / 100 μl of peripheral whole blood).

Separation of human CD3 negative SIT positive lymphocytes (red-filled) from CD3 negative SIT negative lymphocytes (black-dashed) in flow cytometry analysis (intracellular staining) of peripheral whole blood stained using anti-SIT (SIT-01) purified antibody (concentration in sample 9 μg/ml, GAM APC).

Flow cytometry intracellular staining pattern of human peripheral whole blood using anti-SIT (SIT-01) purified antibody (concentration in sample 9 μg/ml, GAM APC).

Western blotting analysis of human SIT using mouse monoclonal antibody SIT-01 on lysates of Molt-4 and HEK-293T cells under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2 µg/ml of mouse anti-SIT monoclonal antibody followed by IRDye800-conjugated anti-mouse secondary antibody. SIT was detected around 36 kDa.

Anti-SIT Purified (clone SIT-01) specificity verification by WB.

The specificity of SIT-01 antibody was assessed by comparing binding signals in HEK293T cells overexpressing the target SIT protein to wild type cells (control) with low level of endogenous protein expression.

Western blotting analysis was performed on whole cell extracts (urea lysis buffer) of transfected and control cells, mixed and heated (100°C, 5 min) with reducing (2-mercaptoethanol) SDS-loading buffer. Samples were resolved using 10% SDS-PAGE gel.

Nitrocellulose membrane blot was probed simultaneously with mouse IgG1 monoclonal antibody SIT-01 (2 µg/ml), and rat IgG2a anti-tubulin monoclonal antibody YOL1/34 (1 µg/ml) used as the loading control. Subclass-specific secondary antibodies IRDye 800CW Goat-anti-Rat IgG (green) and IRDye 680LT Goat-anti-Mouse IgG (red) were used for multiplex fluorescent Western blot detection.

General references:

Hubener C, Mincheva A, Lichter P, Schraven B, Bruyns E: Complete sequence, genomic organization, and chromosomal localization of the human gene encoding the SHP2-interacting transmembrane adaptor protein (SIT). Immunogenetics. 2001 May-Jun;53(4):337-41.
PubMed

Simeoni L, Posevitz V, Kolsch U, Meinert I, Bruyns E, Pfeffer K, Reinhold D, Schraven B: The transmembrane adapter protein SIT regulates thymic development and peripheral T-cell functions. Mol Cell Biol. 2005 Sep;25(17):7557-68.
PubMed

Marie-Cardine A, Kirchgessner H, Bruyns E, Shevchenko A, Mann M, Autschbach F, Ratnofsky S, Meuer S, Schraven B: SHP2-interacting transmembrane adaptor protein (SIT), a novel disulfide-linked dimer regulating human T cell activation. J Exp Med. 1999 Apr 19;189(8):1181-94.
PubMed

Product specific references:

Tedoldi S, Paterson JC, Hansmann ML, Natkunam Y, Rüdiger T, Angelisova P, Du MQ, Roberton H, Roncador G, Sanchez L, Pozzobon M, Masir N, Barry R, Pileri S, Mason DY, Marafioti T, Horejsí V: Transmembrane adaptor molecules: a new category of lymphoid-cell markers. Blood. 2006 Jan 1;107(1):213-21.
PubMed