KDEL Antibody (Rabbit mAb) [C2B2]

Application: Reactivity: Human, Mouse, Rat

Usage Information

Dilution
WB1:1000 IHC1:50 - 1:350 IF1:100 - 1:250 FCM1:10 - 1:100

Application
WB, IHC, IF, FCM

Reactivity
Human, Mouse, Rat

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

Positive Control	Mouse liver tissue; Human liver tissue; Rat kidney tissue; Mouse kidney tissue; Jurkat cells; HeLa cells; Raw264.7 cells; C6 cells
Negative Control

Datasheet & SDS

Biological Description

Specificity
KDEL Antibody (Rabbit mAb) [C2B2] detects endogenous levels of total KDEL protein.

Clone
C2B2

Synonym(s)
Lys Asp Glu Leu

Background
The KDEL retrieval system consists of a short carboxyl-terminal tetrapeptide signal, Lys-Asp-Glu-Leu or a closely related variant such as His-Asp-Glu-Leu or Arg-Asp-Glu-Leu, displayed on the C-terminus of soluble endoplasmic reticulum luminal proteins, and a seven-transmembrane receptor, KDELR, that captures proteins bearing this signal after they escape into the Golgi and returns them to the ER. Crystal structures of the receptor in its apo, ER-resident state and in its ligand-bound, Golgi state reveal a transporter-like architecture structurally related to the PQ-loop family of small-molecule transporters rather than to classical G-protein-coupled receptors, and the receptor undergoes a defined conformational change upon KDEL binding, with movement of transmembrane helix 7 exposing a cytoplasmic lysine motif that is directly recognized by the COPI coat complex, triggering packaging of the receptor-cargo complex into COPI-coated retrograde transport vesicles. Signal capture proceeds through a stepwise relay mechanism rather than a single-step binding event: the free carboxyl terminus of the retrieval signal is initially engaged by receptor residue Arg169, then transferred to Arg5, followed by interaction of the signal's glutamate residue with Arg169, and finally Arg47 engages the carboxyl terminus while the signal's aspartate residue contacts Arg169, progressively drawing the peptide fully into the binding pocket. Selectivity against non-cognate C-terminal sequences is enforced by gatekeeper residues Asp50 and Glu117 positioned at the entrance to this binding pocket, which exclude sequences such as ADEL and DDEL from productive engagement, and mutating both gatekeeper residues simultaneously shifts receptor selectivity to accept these otherwise-excluded variants, directly confirming their proofreading function. Differences in binding affinity among natural signal variants, HDEL binding roughly tenfold more tightly than KDEL, are explained not by the gatekeeper residues but by distinct interactions between the variable fourth position of the signal, histidine, lysine, or arginine, and receptor residue Trp120, giving the KDEL retrieval system a tunable dynamic range capable of efficiently retrieving both low- and high-abundance ER proteins. Once the receptor-cargo complex reaches the neutral pH environment of the ER, the receptor undergoes deprotonation, releasing the retrieval signal and its associated cargo, reversing the conformational change to bury the COPI-binding lysine motif and instead expose an acidic patch that serves as a COPII-binding ER exit signal, allowing the empty receptor to be recycled back to the Golgi.

Background

The KDEL retrieval system consists of a short carboxyl-terminal tetrapeptide signal, Lys-Asp-Glu-Leu or a closely related variant such as His-Asp-Glu-Leu or Arg-Asp-Glu-Leu, displayed on the C-terminus of soluble endoplasmic reticulum luminal proteins, and a seven-transmembrane receptor, KDELR, that captures proteins bearing this signal after they escape into the Golgi and returns them to the ER. Crystal structures of the receptor in its apo, ER-resident state and in its ligand-bound, Golgi state reveal a transporter-like architecture structurally related to the PQ-loop family of small-molecule transporters rather than to classical G-protein-coupled receptors, and the receptor undergoes a defined conformational change upon KDEL binding, with movement of transmembrane helix 7 exposing a cytoplasmic lysine motif that is directly recognized by the COPI coat complex, triggering packaging of the receptor-cargo complex into COPI-coated retrograde transport vesicles. Signal capture proceeds through a stepwise relay mechanism rather than a single-step binding event: the free carboxyl terminus of the retrieval signal is initially engaged by receptor residue Arg169, then transferred to Arg5, followed by interaction of the signal's glutamate residue with Arg169, and finally Arg47 engages the carboxyl terminus while the signal's aspartate residue contacts Arg169, progressively drawing the peptide fully into the binding pocket. Selectivity against non-cognate C-terminal sequences is enforced by gatekeeper residues Asp50 and Glu117 positioned at the entrance to this binding pocket, which exclude sequences such as ADEL and DDEL from productive engagement, and mutating both gatekeeper residues simultaneously shifts receptor selectivity to accept these otherwise-excluded variants, directly confirming their proofreading function. Differences in binding affinity among natural signal variants, HDEL binding roughly tenfold more tightly than KDEL, are explained not by the gatekeeper residues but by distinct interactions between the variable fourth position of the signal, histidine, lysine, or arginine, and receptor residue Trp120, giving the KDEL retrieval system a tunable dynamic range capable of efficiently retrieving both low- and high-abundance ER proteins. Once the receptor-cargo complex reaches the neutral pH environment of the ER, the receptor undergoes deprotonation, releasing the retrieval signal and its associated cargo, reversing the conformational change to bury the COPI-binding lysine motif and instead expose an acidic patch that serves as a COPII-binding ER exit signal, allowing the empty receptor to be recycled back to the Golgi.

References
https://pubmed.ncbi.nlm.nih.gov/30846601/ https://pubmed.ncbi.nlm.nih.gov/34137369/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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%