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Cytotoxic T-Lymphocyte Associated Protein 4 Peptide 2

Antibody: CPTC-CTLA4-2
Background

Catalog Number:

CPTC-CTLA4-2

Target Antigen:

Cytotoxic T-Lymphocyte Associated Protein 4 Peptide 2

Isotype:

IgG

Species:

Rabbit Monoclonal Antibody

Last Updated:

05/04/2023

Antigen Recognition(s):

Peptide, Endogenous

Characterization Data [Compare Characterization Data]
  Affinity Measurement by Biolayer Interferometry
Click to enlarge image The affinity and binding kinetics of CPTC-CTLA4-2 antibody and BSA-conjugated peptide “AMHVAQPAVVLASSR" were measured using biolayer interferometry. BSA-conjugated peptide was covalently immobilized onto AR2G biosensors using standard amine coupling. CPTC-CTLA4-2 antibody, 256 nM, 64 nM, 16 nM, 4 nM, and 1 nM, was used as analyte. Buffer only and biosensors immobilized without BSA-conjugated peptide were used as references for background subtraction. All data was analyzed globally using a 1:2 bivalent model. Click image to enlarge

CPTC-CTLA4-2 Affinity and Kinetics (Bio-Layer Interferometry)

Result: Positive

The affinity and binding kinetics of CPTC-CTLA4-2 antibody and BSA-conjugated peptide “AMHVAQPAVVLASSR" were measured using biolayer interferometry. BSA-conjugated peptide was covalently immobilized onto AR2G biosensors using standard amine coupling. CPTC-CTLA4-2 antibody, 256 nM, 64 nM, 16 nM, 4 nM, and 1 nM, was used as analyte. Buffer only and biosensors immobilized without BSA-conjugated peptide were used as references for background subtraction. All data was analyzed globally using a 1:2 bivalent model.


Characterization SOP Files

  Affinity Measurement by SPR
Click to enlarge image The affinity and binding kinetics of CPTC-CTLA4-2 antibody and BSA-conjugated peptide, “AMHVAQPAVVLASSR”, were measured using surface plasmon resonance. Peptide was amine coupled onto a Series S CM5 biosensor chip. Antibody at 256 nM, 64 nM, 16 nM, 4 nM and 1.0 nM, was used as analyte. Binding data were double-referenced and analyzed globally using a bivalent fitting model. Click image to enlarge

CPTC-CTLA4-2 Affinity and Kinetics (Surface Plasmon Resonance) 

Result: Positive

The affinity and binding kinetics of CPTC-CTLA4-2 antibody and BSA-conjugated peptide, “AMHVAQPAVVLASSR”, were measured using surface plasmon resonance. Peptide was amine coupled onto a Series S CM5 biosensor chip. Antibody at 256 nM, 64 nM, 16 nM, 4 nM and 1.0 nM, was used as analyte. Binding data were double-referenced and analyzed globally using a bivalent fitting model.


Characterization SOP Files

  IHC Tissue
Click to enlarge image Tissue Micro-Array (TMA) core of colon cancer showing membranous staining using Antibody CPTC-CTLA4-2. Titer: 1:500 Click image to enlarge

CPTC-CTLA4-2 IHC Tissue

Result: Positive

Tissue Micro-Array (TMA) core of colon cancer showing membranous staining using Antibody CPTC-CTLA4-2. Titer: 1:500


Characterization SOP Files

  Immuno-MRM
Click to enlarge image Immuno-MRM using CPTC-CTLA4-2 as capture antibody against the synthetic peptide AMHVAQPAVVLASSR. Antibody CPTC-CTLA4-2 captures the synthetic peptide. Click image to enlarge

CPTC-CTLA4-2 iMRM (synthetic peptide)

Result: Positive

Immuno-MRM using CPTC-CTLA4-2 as capture antibody against the synthetic peptide AMHVAQPAVVLASSR. Antibody CPTC-CTLA4-2 captures the synthetic peptide.


Characterization SOP Files

  Immunofluorescence

CPTC-CTLA4-2 Immunofluorescence

Result: Negative

Immunofluorescence staining using CPTC-CTLA4-2 as primary antibody against MCF7 cells shows no localization of CTLA4 protein.


Characterization SOP Files

  Indirect ELISA
Click to enlarge image Indirect ELISA using CPTC-CTLA4-2 as primary antibody against BSA conjugated peptide "AMHVAQPAVVLASSR", coated on the plate. Peptide was detected using HRP conjugated goat anti-rabbit secondary antibody and tetramethylbenzidine (TMB). Click image to enlarge

CPTC-CTLA4-2 Indirect ELISA

Result: Positive

Indirect ELISA using CPTC-CTLA4-2 as primary antibody against BSA conjugated peptide "AMHVAQPAVVLASSR", coated on the plate. Peptide was detected using HRP conjugated goat anti-rabbit secondary antibody and tetramethylbenzidine (TMB).


Characterization SOP Files

  NCI 60 Protein Array

CPTC-CTLA4-2 NCI 60 Protein Array

Result: Negative

This antibody is not suitable for use in a Reverse Phase Protein Array format as described in SOP M-105.


Characterization SOP Files

  Western Blot - Recombinant Protein or Peptide

CPTC-CTLA4-2 Western Blot (Recombinant Protein)

Result: Negative

Western blot using CPTC-CTLA4-2 as primary antibody against human cytotoxic T-lymphocyte-associated protein 4 (CTLA4), transcript variant 1. The expected molecular weight is 16.0 kDa.


Characterization SOP Files

  Western Blot - Recombinant Protein or Peptide
Click to enlarge image Automated western blot using CPTC-CTLA4-2 as primary antibody against recombinant CTLA4 protein. Protein molecular weight is about 25 KDa, but the protein is glycosylated and runs at a higher molecular weight. The antibody cannot recognize recombinant CTLA4. Click image to enlarge

CPTC-CTLA4-2 Automated Western Blot (Recombinant Protein)

Result: Negative

Automated western blot using CPTC-CTLA4-2 as primary antibody against recombinant CTLA4 protein. Protein molecular weight is about 25 KDa, but the protein is glycosylated and runs at a higher molecular weight. The antibody cannot recognize recombinant CTLA4.


Characterization SOP Files

  Western Blot - Tissue or Cell Lysate
Click to enlarge image Western blot using CPTC-CTLA4-2 as primary antibody against whole cell lysates CCRF-CEM (lane 2), HeLa (lane 3), Jurkat (lane 4), K-562 (lane 5), and MCF7 (lane 6). Molecular weight standards are also included. The expected molecular weights are 24.7 kDa, 6.6 kDa, 6.7 kDa, 8.9 kDa, and 19.1 kDa. HeLa is presumed positive. MCF7 is positive. All other cell lines are negative. CTLA4 is subject to glycosylation which can affect its migration in electrophoresis. This can make the target appear as a higher molecular weight protein. Click image to enlarge

CPTC-CTLA4-2 Western Blot (Cell Lysates)

Result: Positive

Western blot using CPTC-CTLA4-2 as primary antibody against whole cell lysates CCRF-CEM (lane 2), HeLa (lane 3), Jurkat (lane 4), K-562 (lane 5), and MCF7 (lane 6). Molecular weight standards are also included. The expected molecular weights are 24.7 kDa, 6.6 kDa, 6.7 kDa, 8.9 kDa, and 19.1 kDa. HeLa is presumed positive. MCF7 is positive. All other cell lines are negative. CTLA4 is subject to glycosylation which can affect its migration in electrophoresis. This can make the target appear as a higher molecular weight protein.


Characterization SOP Files

  Western Blot - Tissue or Cell Lysate
Click to enlarge image Automated western blot using CPTC-CTLA4-2 as primary antibody against whole lysates of cell lines CCRF-CEM, HeLa, Jurkat, K-562 and MCF7. Protein molecular weight is about 25 KDa, but the protein is glycosylated and runs at a higher molecular weight. The antibody recognizes CTLA4 in MCF7 lysate. Loading controls were run with anti-GAPDH antibody. Click image to enlarge

CPTC-CTLA4-2 Automated Western Blot (Cell Lysates)

Result: Positive

Automated western blot using CPTC-CTLA4-2 as primary antibody against whole lysates of cell lines CCRF-CEM, HeLa, Jurkat, K-562 and MCF7. Protein molecular weight is about 25 KDa, but the protein is glycosylated and runs at a higher molecular weight. The antibody recognizes CTLA4 in MCF7 lysate. Loading controls were run with anti-GAPDH antibody.


Characterization SOP Files

  Western Blot - Tissue or Cell Lysate
Click to enlarge image Single cell western blot using CPTC-CTLA4-2 as a primary antibody against cell lysates. Relative expression of total CTLA4 in MCF7 cells (A). Percentage of cells that express CTLA4 (B). Average expression of CTLA4 protein per cell (C). All data is normalized to β-tubulin expression. Click image to enlarge

CPTC-CTLA4-2 Single Cell Western Blot (Cell Lysate)

Result: Positive

Single cell western blot using CPTC-CTLA4-2 as a primary antibody against cell lysates. Relative expression of total CTLA4 in MCF7 cells (A). Percentage of cells that express CTLA4 (B). Average expression of CTLA4 protein per cell (C). All data is normalized to β-tubulin expression.


Characterization SOP Files

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Antigen: Cytotoxic T-Lymphocyte Associated Protein 4 Peptide 2
Background

NCI Identification Number:

00517

Antigen Name:

Cytotoxic T-Lymphocyte Associated Protein 4 Peptide 2

CPTC Name:

CPTC-CTLA4 Peptide 2

Aliases:

Cytotoxic T-Lymphocyte Associated Protein 4; CTLA-4; CD152; GSE; CD; Insulin-Dependent Diabetes Mellitus 12; Cytotoxic T-Lymphocyte Protein 4; Celiac Disease 3; CELIAC3; IDDM12; Ligand And Transmembrane Spliced Cytotoxic T Lymphocyte Associated Antigen 4; Cytotoxic T Lymphocyte Associated Antigen 4 Short Spliced Form; Cytotoxic T-Lymphocyte-Associated Serine Esterase-4; Cytotoxic T-Lymphocyte-Associated Antigen 4; CD152 Antigen; ALPS5; GRD4

Function:

This gene is a member of the immunoglobulin superfamily and encodes a protein which transmits an inhibitory signal to T cells. The protein contains a V domain, a transmembrane domain, and a cytoplasmic tail. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. The membrane-bound isoform functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. Mutations in this gene have been associated with insulin-dependent diabetes mellitus, Graves disease, Hashimoto thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, and other autoimmune diseases.CTLA4 (Cytotoxic T-Lymphocyte Associated Protein 4) is a Protein Coding gene. Diseases associated with CTLA4 include Autoimmune Lymphoproliferative Syndrome, Type V and Celiac Disease 3. Among its related pathways are TCR Signaling (Qiagen) and T cell receptor signaling pathway. An important paralog of this gene is CD28.Inhibitory receptor acting as a major negative regulator of T-cell responses. The affinity of CTLA4 for its natural B7 family ligands, CD80 and CD86, is considerably stronger than the affinity of their cognate stimulatory coreceptor CD28.

Chromosomal Localization:

2q33.2

Accession Number:

NP_005205.2

UniProt Accession Number:

P16410

DNA Source:

N/A

Immunogen:

Synthetic Peptide

Vector Name:

N/A

Extinction Coefficient:

Buffers:

Expressed Sequence:

AMHVAQPAVVLASSR

Native Sequence:

Calculated Isoelectric Point:

Molecular Weight:

1650

Last Updated:

05/31/2022

Links

Characterization Data

SOPs

No SOPs available.

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