ESD4 Antibody

Introduction to ESD4 Antibody

The ESD4 antibody targets esterase D (ESD), also known as S-formylglutathione hydrolase, a protein of approximately 31 kDa . Research indicates that ESD4 plays a crucial role in various cellular processes, particularly in plant immunity and ABA signaling during seed germination .

ESD4 Antibody Specificity and Applications

The ESD4 antibody, such as the [EPR8447] clone, is utilized in western blotting to detect ESD protein in cell lysates . It has been shown to recognize ESD in various cell lines, including HEK-293T, K562, Jurkat, Caco-2, and HeLa . The antibody can distinguish between wild-type and ESD knockout cell lines, confirming its specificity for the ESD protein .
Syndecan-4 Antibody (5G9) is a mouse monoclonal IgG2a kappa light chain antibody engineered to detect Syndecan-4, also known as amphiglycan or ryudocan, across mouse, rat, and human species .

Role in Plant Immunity

In Arabidopsis thaliana, ESD4 coordinates with NUA to deSUMOylate TPR1, a process that positively regulates plant immunity . The deSUMOylation of TPR1 by ESD4 is specific, as another SUMO protease, OTS2, does not have the same effect .

Impact on ABA Signaling

Studies have demonstrated that nua and esd4 mutants are hypersensitive to abscisic acid (ABA) during seed germination and seedling growth, implying that NUA and ESD4 negatively regulate ABA signaling . The esd4-3 mutants accumulate more SUMO conjugates and exhibit less free SUMO compared to nua-3 mutants, suggesting that ESD4 plays a more significant role in deSUMOylation .

Research Findings

Experimental Protocols

Western Blotting Protocol

1. Prepare cell lysates from various cell lines (e.g., HEK-293T, K562, Jurkat) using standard lysis buffer.

2. Determine protein concentrations using a BCA assay or similar method.

3. Load equal amounts of protein (e.g., 10-20 µg) per lane on an SDS-PAGE gel.

4. Perform electrophoresis to separate proteins by size.

5. Transfer proteins from the gel to a PVDF or nitrocellulose membrane.

6. Block the membrane with 5% non-fat milk or BSA in TBST buffer to prevent non-specific binding.

7. Incubate the membrane with the primary ESD4 antibody (e.g., Anti-ESD [EPR8447] at 1/1000 - 1/10000 dilution) overnight at 4°C.

8. Wash the membrane three times with TBST buffer.

9. Incubate the membrane with an appropriate HRP-conjugated secondary antibody (e.g., Goat anti-Rabbit HRP at 1/2000 dilution) for 1 hour at room temperature.

10. Wash the membrane three times with TBST buffer.

11. Detect protein bands using an enhanced chemiluminescence (ECL) substrate and a ChemiDoc imaging system.