TPN Antibody (Rabbit mAb) [N20A4]

Application: Reactivity: Mouse, Human

Usage Information

Dilution
WB1:1000 IHC1:500 IF1:500 FCM1:500

Application
WB, IHC, IF, FCM

Reactivity
Mouse, Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
48 kDa 48 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Mouse spleen tissue; Human liver tissue; Human lung tissue; Human colon carcinoma tissue; Human lung adenocarcinoma tissue; Human colon tissue; HeLa cells (IFNγ, 100 ng/ml, 16 h); B16-F10 cells (IFNγ, 100 ng/ml, 16 h); RAW264.7 cells; HeLa cells; Jurkat cells; A431 cells
Negative Control	B16-F10

Datasheet & SDS

Biological Description

Specificity
TPN Antibody (Rabbit mAb) [N20A4] detects endogenous levels of total TPN protein.

Clone
N20A4

Synonym(s)
NGS17, TAPA, TAPBP, Tapasin, TPN, TPSN, NGS-17, TAP-associated protein, TAP-binding protein

Background
Tapasin is an endoplasmic reticulum-resident type I transmembrane glycoprotein and functions as the central catalytic chaperone within the peptide-loading complex, a multiprotein assembly built from the TAP1/TAP2 heterodimeric peptide transporter, tapasin, the thiol oxidoreductase ERp57, and the sugar-binding chaperone calreticulin, coordinating selection and loading of antigenic peptides onto MHC class I molecules. Tapasin physically bridges TAP to newly assembled MHC I heavy chain–beta2-microglobulin heterodimers, and this bridging interaction is required for assembly and function of multimeric MHC class I-TAP complexes, positioning peptides delivered into the endoplasmic reticulum lumen by TAP directly adjacent to the MHC I peptide-binding groove for loading. The tapasin-stabilized, peptide-receptive conformation of MHC I is characterized by distortion of the peptide-binding groove and destabilization of the noncovalent interaction between the heavy chain and beta2-microglobulin, with coordinated movements of the membrane-proximal immunoglobulin-like domains of tapasin, MHC I, and beta2-microglobulin accompanying transition into a state primed to release low-affinity peptide. Tapasin exerts a continuous mechanical force on the MHC I peptide-binding groove that acts to open it, while bound peptides exert an opposing force determined by their binding affinity; low-affinity peptides generate insufficient opposing force to keep the groove closed and are released rapidly, whereas high-affinity peptides overcome tapasin's opening force, stabilize the closed groove conformation, and trigger their own retention along with dissociation of the surrounding peptide-loading complex. Through this force-based competition, tapasin iteratively samples and discards successive low-affinity peptides until a peptide of sufficient affinity is captured, skewing the final MHC I-bound peptide repertoire toward high-affinity, immunodominant epitopes rather than presenting the first peptide encountered. Once a high-affinity peptide is stably bound, the peptide-loaded MHC I heterodimer dissociates from tapasin and the peptide-loading complex and proceeds through the secretory pathway to the cell surface for recognition by CD8-positive T cells and natural killer cell receptors. Different MHC I allotypes vary in the degree to which they depend on tapasin for effective peptide selection, and loss of tapasin function destabilizes cell-surface MHC I expression, connecting tapasin's peptide-editing mechanism directly to immune recognition of infected or malignantly transformed cells and to antigen-presentation-modulating therapeutic strategies.

Background

Tapasin is an endoplasmic reticulum-resident type I transmembrane glycoprotein and functions as the central catalytic chaperone within the peptide-loading complex, a multiprotein assembly built from the TAP1/TAP2 heterodimeric peptide transporter, tapasin, the thiol oxidoreductase ERp57, and the sugar-binding chaperone calreticulin, coordinating selection and loading of antigenic peptides onto MHC class I molecules. Tapasin physically bridges TAP to newly assembled MHC I heavy chain–beta2-microglobulin heterodimers, and this bridging interaction is required for assembly and function of multimeric MHC class I-TAP complexes, positioning peptides delivered into the endoplasmic reticulum lumen by TAP directly adjacent to the MHC I peptide-binding groove for loading. The tapasin-stabilized, peptide-receptive conformation of MHC I is characterized by distortion of the peptide-binding groove and destabilization of the noncovalent interaction between the heavy chain and beta2-microglobulin, with coordinated movements of the membrane-proximal immunoglobulin-like domains of tapasin, MHC I, and beta2-microglobulin accompanying transition into a state primed to release low-affinity peptide. Tapasin exerts a continuous mechanical force on the MHC I peptide-binding groove that acts to open it, while bound peptides exert an opposing force determined by their binding affinity; low-affinity peptides generate insufficient opposing force to keep the groove closed and are released rapidly, whereas high-affinity peptides overcome tapasin's opening force, stabilize the closed groove conformation, and trigger their own retention along with dissociation of the surrounding peptide-loading complex. Through this force-based competition, tapasin iteratively samples and discards successive low-affinity peptides until a peptide of sufficient affinity is captured, skewing the final MHC I-bound peptide repertoire toward high-affinity, immunodominant epitopes rather than presenting the first peptide encountered. Once a high-affinity peptide is stably bound, the peptide-loaded MHC I heterodimer dissociates from tapasin and the peptide-loading complex and proceeds through the secretory pathway to the cell surface for recognition by CD8-positive T cells and natural killer cell receptors. Different MHC I allotypes vary in the degree to which they depend on tapasin for effective peptide selection, and loss of tapasin function destabilizes cell-surface MHC I expression, connecting tapasin's peptide-editing mechanism directly to immune recognition of infected or malignantly transformed cells and to antigen-presentation-modulating therapeutic strategies.

References
https://pubmed.ncbi.nlm.nih.gov/36115831/ https://pubmed.ncbi.nlm.nih.gov/26754481/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%