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DFNA5/GSDME Rabbit mAb

Catalog No.: F1701

Application: Reactivity: Human, Mouse, Rat

Lane 1: SH-SY5Y

Customer Reviews

Experiment Essentials

This antibody can recognize the full-chain DFNA5/GSDME and the N-terminal part after cleavage of DFNA5/GSDME.

DFNA5/GSDME protein is only expressed in some cells\tissues, and the expression levels vary. When performing WB experiments, it is recommended to set up positive and negative control groups to optimize the experimental system.
If you need to detect the N-terminal fragment of DFNA5/GSDME protein in cell samples, it is recommended to induction treatment (such as SH-SY5Y, 60uM etoposide, 14h treatment to increase the expression abundance of the N-terminal fragment of DFNA5/GSDME protein).

Usage Information

Dilution
WB1:1000 IP1:30 FCM1:60

Application
WB, IP, FCM

Reactivity
Human, Mouse, Rat

Source
Rabbit

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
54 kDa 37 kDa, 55 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight.

Positive Control	Mouse brain; Mouse liver; SH-SY5Y; EMT6; SGC-7901; HEK-293 (transfected with DDDDK tagged DFNA5/GSDME)
Negative Control	HEK-293

Expression & Treatment Conditions

Sample	Treatment Conditions
MCF-7	Low Expression
Click to view more sample data

^*For predicted expression levels of this protein in various human-derived cells and tissues, please refer to: http://www.proteinatlas.org

Sample	Treatment Conditions

Exprimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 962. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

962. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
DFNA5/GSDME Rabbit mAb recognizes endogenous levels of total DFNA5/GSDME protein.

Subcellular Location
Cell membrane, Cytoplasm, Membrane

Uniprot ID
O60443

Clone
G9F19

Synonym(s)
cleaved N-terminal DFNA5,DFNA5/GSDME,Gasdermin E

Background
Gasdermin E (GSDME), also known as deafness autosomal dominant 5 (DFNA5), was initially identified as a gene linked to an autosomal dominant form of inherited hearing loss. Members of the gasdermin family are known for their role in mediating inflammatory cell death through their pore-forming ability. Cleavage of gasdermins between domains enables the amino-terminal domain to bind to membrane phospholipids and oligomerize, forming a pore on the plasma membrane. This process results in rapid cell swelling, the formation of large bubbles on the plasma membrane, and eventual cell lysis, a form of programmed necrosis known as pyroptosis. Gasdermin E (GSDME) is distinct among the gasdermins because its activation requires cleavage by caspase-3, an enzyme involved in the apoptosis of tumor cells. In many types of tumor cells, GSDME is silenced due to hypermethylation of its promoter region. However, when expressed, GSDME can induce tumor cell death and boost antitumor immunity. In cancer biology, DFNA5 acts as a tumor suppressor, inhibiting colony formation and cell proliferation in gastric cancer, melanoma, and colorectal cancer cells, as well as reducing the aggressive behavior of breast cancer.

Background

Gasdermin E (GSDME), also known as deafness autosomal dominant 5 (DFNA5), was initially identified as a gene linked to an autosomal dominant form of inherited hearing loss. Members of the gasdermin family are known for their role in mediating inflammatory cell death through their pore-forming ability. Cleavage of gasdermins between domains enables the amino-terminal domain to bind to membrane phospholipids and oligomerize, forming a pore on the plasma membrane. This process results in rapid cell swelling, the formation of large bubbles on the plasma membrane, and eventual cell lysis, a form of programmed necrosis known as pyroptosis. Gasdermin E (GSDME) is distinct among the gasdermins because its activation requires cleavage by caspase-3, an enzyme involved in the apoptosis of tumor cells. In many types of tumor cells, GSDME is silenced due to hypermethylation of its promoter region. However, when expressed, GSDME can induce tumor cell death and boost antitumor immunity. In cancer biology, DFNA5 acts as a tumor suppressor, inhibiting colony formation and cell proliferation in gastric cancer, melanoma, and colorectal cancer cells, as well as reducing the aggressive behavior of breast cancer.

References
https://pubmed.ncbi.nlm.nih.gov/34433433/ https://pubmed.ncbi.nlm.nih.gov/35292781/

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

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

* Indicates a Required Field

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%