EuroMAbNet: European Monoclonal Antibodies NetworkEuroMAbNet. European Monoclonal Antibodies Network homepage

Menu

Antibody Identity card

Antibody information [TRAT1 ]

MARTIN604A

  • Clone name MARTIN604A
  • Description Rat monoclonal
  • Antigen used HIS-GST-TRAT1 (29-186end)
  • Epitope unkown
  • Isotype IgG2a, K
  • Confirmed species reactivity Human
  • Ab used TRAT1 CNIO (clone MARTIN604A)
APPLICATIONRecommended concentrationStatusProtocol
Western blotting (WB)No dilution (neat tissue culture supernatant)WorkingWestern Blotting (WB)
Immunocytochemistry (ICC)No dilution (neat tissue culture supernatant)WorkingICC in frozen tissue and cytospin preparation
Immunohistochemistry (IHC-P)1:30 (neat tissue culture supernatant)WorkingBOND-MAX automated immunohistochemistry
Immunoflourescence (IF)1:2 (neat tissue culture supernatant)Immunofluorescence staining
Flow cytometry (FC)30ul supernatant/100ul PBSWorkingFlow cytometry
IHC-P SpeciesNot tested

Ab ID Card application validation / characterisation

WB Validation
Over-expression/cross reactivity
TRAT1 antibody WB Validation: Over-expression/cross reactivity
To confirm the specificity of the antibody, Western blotting of the MARTIN604A mAb was performed using cell extracts of HEK293T cells overexpressing human TRAT1-GFP and AID.A single band of about 50kDa was found only in the TRAT1 transfected cells. Anti-vinculin was used as the loading control.

Show image in a new window

Gene inactivation
TRAT1 antibody WB Validation: Gene inactivation
The specificity of the MARTIN604A mAb for the endogenous TRAT1 protein was confirmed by WB using cell extracts of the MOLT4 cell line before (MOLT4 ) and after TRAT1 gene inactivation (TRAT1 KO MOLT4) using CRISPR-Cas9 technology. A 30 kDa band was present only in the MOLT4 cell extract. Anti-vinculin was used as loading control.

Show image in a new window

WB Characterisation
Endogenous expression
TRAT1 antibody WB Characterisation: Endogenous expression
WB using the MARTIN604A mAb showed 30 kDa and 50kDa bands of TRAT1 protein in tonsil tissue extract as well as in extracts of the JURKAT, MOLT4, MOLT16 (acute lymphoblastic leukemia), and LP1 (plasma cell myeloma) cell lines. No bands were seen in the kidney tissue extract or for L428, DEV (Hodgkin's lymphoma), and U266 (myeloma) cell extracts. Anti-vinculin was used as a loading control.

Show image in a new window

ICC / ICC-P Validation
Over-expression/cross reactivity
TRAT1 antibody ICC Validation: Over-expression/cross reactivity
Immunocytochemistry (ICC) of the MARTIN604A mAb was performed on frozen cytospin preparations of GFP-tagged human TRAT1 and human LAX1 proteins expressed in HEK293T cells. Labelling with the MARTIN604A mAb was present in the HEK-TRAT1-GFP transfectants but not in the negative control HEK-LAX1-GFP cells. Labelling with anti-GFP was used to confirm the efficiency of transfection.

Show image in a new window

Gene inactivation
TRAT1 antibody ICC Validation: Gene inactivation
The specificity of the MARTIN604A mAb for the endogenous TRAT1 protein was confirmed by immunoperoxidase (IHC-P) staining of sections from formalin fixed paraffin embedded cell pellets of the MOLT4 cell line before (MOLT4 WT) and after (TRAT1 KO MOLT4) gene inactivation using CRISPR-Cas9 technology. TRAT1 membrane expression was observed in wild type MOLT4 cells, while no staining was observed in the TRAT1 KO MOLT4 cell line.

Show image in a new window

IHC-P Characterisation
Endogenous expression
TRAT1 antibody IHC-P Characterisation: Endogenous expression
In the tonsil, MARTIN604A is expressed in T lymphocytes located both within and outside the germinal center. In the spleen, TRAT1 is predominantly observed in T cells surrounding the arterioles in the red pulp while in the thymus its expression is detected in both the medulla and cortex. Additionally, a population of positive cells is also present in the bone marrow.

Show image in a new window

IF Characterisation
Endogenous expression
TRAT1 antibody IF Characterisation: Endogenous expression
Triple immunofluorescence on tonsil paraffin sections was performed using antibodies MARTIN604A (labeled in red), CD8 (labeled in pink), CD3 (labeled in green) and DAPI (labeled in blue).

Show image in a new window

FC Validation
Over-expression/cross reactivity
TRAT1 antibody FC Validation: Over-expression/cross reactivity
Overexpression of the TRAT1 protein was confirmed by flow cytometry using the mAb MARTIN604A. HEK293T cells transfected with TRAT1 served as a positive control (light blue), while HEK293T cells transfected with AID were used as a negative control (pink). Control experiments using only the secondary antibody showed no staining for HEK AID cells (dashed pink line) or HEK TRAT1 cells (dashed blue line).

Show image in a new window

Gene inactivation
TRAT1 antibody FC Validation: Gene inactivation
The specificity of the MARTIN604A mAb for endogenous TRAT1 protein was confirmed by flow cytometry using the MOLT4 cell line before (MOLT4, light blue) and after TRAT1 gene inactivation (TRAT1 KO MOLT4, pink) via CRISPR-Cas9. Control experiments using only the secondary antibody showed no staining for TRAT1 KO MOLT4 cells (dashed pink line) or MOLT4 cells (dashed light blue line).

Show image in a new window

FC Characterisation
Endogenous expression
TRAT1 antibody FC Characterisation: Endogenous expression
Flow cytometry using the MARTIN604A mAb demonstrated endogenous TRAT1 protein expression in MOLT4 cells (light blue) but not in U266 cells (pink). Control experiments using only the secondary antibody showed no staining for U266 cells (dashed pink line) or MOLT4 cells (dashed light blue line).

Show image in a new window

IHC-P Domestic species
Not tested
IHC-P Wild species
Not tested
← Back to TRAT1 protein