Gasdermin-D (GSDMD）

Structure of the GSDMD Target Protein

Figure 1: Structure of the GSDMD Target Protein

Introduction to GSDMD

Protein Function

GSDMD is the executioner of pyroptosis; the full-length protein is inactive before cleavage and plays a crucial role in host defense against pathogen infection and danger signals.
GSDMD generates an active N-terminal fragment of approximately 32 kDa through cleavage. This fragment binds to the membrane and forms pores, thereby triggering pyroptosis.
The N-terminal fragment of GSDMD binds to the inner leaflet lipids of the plasma membrane, forming pores with a diameter of 10-15 nm, releasing mature IL-1β and triggering pyroptosis.
The N-terminal fragment of GSDMD is released into the extracellular environment from pyroptotic cells and rapidly binds to and kills Gram-negative and Gram-positive bacteria.
The N-terminal fragment of GSDMD does not damage the body's cells due to its specific lipid-binding properties and does not disrupt the plasma membrane from the outside.
The N-terminal fragment of GSDMD does not bind to unphosphorylated phosphatidylinositol, phosphatidylethanolamine, or phosphatidylcholine.

Protein Characteristics

The active N-terminal fragment of GSDMD typically requires induction to be produced, making it essential to induce the samples.

Protein Expression

Expressed in esophageal basal cells and the isthmus/neck, pits, and stomach glands. Preferentially expressed in differentiated cells.

Protein Localization

GSDMD: Cytoplasm, inflammasome.
GSDMD-N terminal: Plasma membrane, extracellular space, or secretory area.

: GSDMD IHC Experiment Results. Anti-GSDMD antibody [EPR20859] (ab219800). Wild type mouse small intestine (A) and GSDMD KO mouse small intestine (B).

Figure 2: GSDMD IHC Experiment Results. Anti-GSDMD antibody [EPR20859] (ab219800). Wild type mouse small intestine (A) and GSDMD KO mouse small intestine (B).

Isoforms & Post-Translational Modifications

Human (P57764): 53 kDa (predicted)
Mouse (Q9D8T2): 53 kDa (predicted)
Rat (A0A096MJ11): 53 kDa (predicted)
GSDMD is cleaved at Asp-275, which releases autoinhibition and initiates pyroptosis. Cleavage at Asp-87 results in the loss of GSDMD's ability to mediate pyroptosis.
GSDMD undergoes succinylation at Cys-191, which activates pyroptosis.

WB Experiment Tips

Precautions

GSDMD exists in multiple forms, including the ~53 kDa GSDMD precursor, the ~31 kDa cleaved GSDMD-N, and the ~22 kDa cleaved GSDMD-C. Select the appropriate antibody based on your detection needs.
GSDMD is cleaved by inflammatory protease caspases in response to typical and atypical inflammasome activators, forming GSDMD-N. In most cases, the precursor protein can be detected without drug treatment; however, samples must be treated to detect the GSDMD-N fragment (e.g., THP-1 cells treated with 500 ng/mL EprI for 2 hours). We recommend using positive and negative controls.
The expression level of GSDMD may vary across different tissues and cells, so it is advisable to set up positive and negative controls.
Tissue samples are more complex than cell samples and may show non-specific bands.

Positive Control

GSDMD: Whole cell lysates of liver or lung from WT mice.
GSDMD: Whole cell lysates of RAW 264.7 or NIH/3T3 cells.
GSDMD-N: Whole cell lysates of THP-1 cells treated with 500 ng/ml EprI for 2 hours.

Negative Control (low or no expression)

GSDMD-N: Untreated whole cell lysates of THP-1 cells.

Example Results

WB - Anti-GSDMD Antibody [EPR20859] (ab219800).

Figure 3: WB - Anti-GSDMD Antibody [EPR20859] (ab219800).

Lane 1: Whole cell lysate of liver from wild type mouse
Lane 2: Whole cell lysate of liver from GSDMD knockout mouse
Lane 3: Whole cell lysate of lung from wild type mouse
Lane 4: Whole cell lysate of lung from GSDMD knockout mouse

Predicted band size: 53 kDa
Detected band size: 53 kDa

This antibody shows strong reactivity with GSDMD, with some weak protein bands.

WB - Anti-cleaved N-terminal GSDMD Antibody [EPR20829-408] (ab215203).

Figure 4: WB - Anti-cleaved N-terminal GSDMD Antibody [EPR20829-408] (ab215203).

Lane 1: Untreated THP-1 whole cell lysate
Lane 2: THP-1 whole cell lysate treated with 500 ng/ml EprI for 2 hours
Blocking/Dilution buffer: 5% NFDM/TBST
Secondary Antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051), diluted 1/100000

Predicted band size: 53 kDa
Detected band size: 31 kDa

Key Control Points

Along with the usual considerations, be sure to focus on the following key control points during the experiment:

Sample Preparation:

Add a cocktail of protease inhibitors to prevent target protein degradation.
Keep samples on ice throughout the entire preparation process.
Determine the total protein concentration using Bradford, Lowry, or BCA assays.
Set up positive and negative controls.

Electrophoresis:

Load at least 20 μg of total protein for electrophoresis.

Transfer:

We recommend staining the membrane with Ponceau S after transfer to confirm successful transfer (ensure Ponceau S is thoroughly washed off if fluorescent detection is used).

Antibody Incubation:

Select the appropriate working concentration of antibodies based on the product datasheet.