DFNA5/GSDME Antibody [G9F19]

Catalog No.: F1701

異なるサンプルでの発現パターンについては、“発現量と処理条件の例表 ”を参照してください。

Application: Reactivity: Human, Mouse, Rat

Lane 1: SH-SY5Y

サイズ（液体）	価格(税別)	在庫状況
20uL	JPY 24800	国内在庫なし(納期7~10日)
100uL	JPY 60800	国内在庫なし(納期7~10日)
100uL*2	JPY 91200	お問い合わせ

代表番号: 045-509-1970\|電子メール：sales@selleck.co.jp よく尋ねられる質問

カスタマーフィードバック(3)

キーポイント

この抗体は、DFNA5/GSDME の完全鎖と、DFNA5/GSDME 切断後の N 末端部分を認識できます。

DFNA5/GSDMEタンパク質は一部の細胞／組織でのみ発現しており、発現レベルは変動します。WB実験を行う際には、実験系を最適化するために陽性対照群と陰性対照群を設定することをお勧めします。
細胞サンプル中のDFNA5/GSDMEタンパク質のN末端断片を検出する必要がある場合は、誘導処理（SH-SY5Y、60uMエトポシド、14時間処理など）を実施して、DFNA5/GSDMEタンパク質のN末端断片の発現量を増加させることをお勧めします。

使用情報

Dilution
WB1:1000 IP1:30 FCM1:60

Application
WB, IP, FCM

Source
Rabbit Monoclonal Antibody

Reactivity
Human, Mouse, Rat

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
^*なぜ予測分子量と実際の分子量が異なるのか？下記の原因により、実際の分子量が予測と異なる：タンパク質の翻訳後修飾（リン酸化/糖鎖付加），スプライシングバリアント，イソフォーム，相対的な電荷，ポリマー。

ポジティブコントロール	Mouse brain; Mouse liver; SH-SY5Y; EMT6; SGC-7901; HEK-293 (transfected with DDDDK tagged DFNA5/GSDME)
ネガティブコントロール	HEK-293

サンプル処理データの例

サンプル	処理状況
MCF-7	Low Expression
クリックして、さらに多くのサンプルデータを表示

^*異なるヒト由来細胞や組織における発現量の予測については、以下をご参照ください: http://www.proteinatlas.org

サンプル	処理状況

プロトコール

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

生物学的記述

Specificity
DFNA5/GSDME Antibody [G9F19] recognizes endogenous levels of total DFNA5/GSDME protein.

タンパク質の局在
細胞膜、細胞質、細胞内膜系

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
[1] Liu Z, et al. BMC Cancer. 2021 Aug 25;21(1):951. [2] LV J , et al. Nat Cell Biol. 2022 Mar;24(3):364-372.

PVDFメンブレン孔径参考表

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

技術サポート

ストックの作り方、阻害剤の保管方法、細胞実験や動物実験の際に注意すべき点など、製品を取扱う時に問い合わせが多かった質問に対しては取扱説明書でお答えしています。

Handling Instructions

他に質問がある場合は、お気軽にお問い合わせください。

* 必須

* 大学・企業名 大学・企業名を記入してください

* 名前 名前を記入してください

* 電子メール 電子メール・アドレスを記入してください 有効なメールアドレスを入力してください

電話番号

* 内容 お問い合わせ内容をご入力ください

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%