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특이성	Na+/H+ Exchanger-1 Antibody [K9D3]는 총 Na+/H+ Exchanger-1 단백질의 내인성 수준을 검출합니다.
배경	Na+/H+ Exchanger-1 (NHE1, SLC9A1)은 세포 내 pH (pHi) 항상성 조절에 핵심적인 역할을 하는 유비쿼터스하게 발현되는 통합 막 역수송체입니다. NHE1은 N-말단과 C-말단이 모두 세포질을 향하는 12개의 막관통 나선(TMHs)으로 구성됩니다. 주요 수송 세그먼트에는 TM IV, VII, IX가 포함되며, 재진입 루프 IL2 및 IL4와 구조적 안정성을 부여하는 세포외 루프 5 (EL5)의 글리코실화 부위도 있습니다. 확장된 약 315개 잔기의 C-말단 세포질 도메인에는 수많은 세린/트레오닌 인산화 부위와 에즈린 결합 모티프 (잔기 553–564)가 포함되어 NHE1을 액틴 세포골격에 고정시킵니다. NHE1은 세포 내 산성화에 의해 알로스테릭하게 활성화되며, 이는 비수송 조절 부위 (힐 계수 ~3)에 양성자 결합을 촉진하여 박테리아 NhaA 수송체와 유사하게 나선 기울기 및 물로 채워진 접근 경로를 통해 내부 지향 상태에서 외부 지향 상태로의 구조적 변화를 유발합니다. 이는 직접적인 에너지 입력 없이 막관통 Na+ 기울기를 구동력으로 사용하여 세포외 Na+ (Km 5–50 mM)와 세포내 H+를 1:1의 화학량론으로 전기발생적으로 교환합니다. 이를 통해 산증 후 pHi의 빠른 회복, Na+ 유입을 통한 세포 부피 조절, ERM-액틴 상호작용을 통한 라멜리포디움 돌출의 스캐폴딩이 가능합니다. 호르몬 및 성장 인자 신호는 NHERF1, ERK, PKA 경로를 통해 C-말단 세린 (예: S703, Thr653)을 인산화하여 pHi 설정점을 알칼리성 쪽으로 이동시킬 수 있습니다. 병리학적으로, 허혈-재관류 손상 시 산성-활성화된 NHE1은 역방향 NCX 활동을 통해 세포독성 Na+/Ca2+ 과부하를 촉진하여 심근경색을 악화시킵니다. 만성 NHE1 상향 조절은 또한 주변 세포 알칼리화 및 기질 리모델링을 통한 종양 침습 및 심장 비대증과 관련이 있습니다.

특이성

Na+/H+ Exchanger-1 Antibody [K9D3]는 총 Na+/H+ Exchanger-1 단백질의 내인성 수준을 검출합니다.

배경

Na+/H+ Exchanger-1 (NHE1, SLC9A1)은 세포 내 pH (pHi) 항상성 조절에 핵심적인 역할을 하는 유비쿼터스하게 발현되는 통합 막 역수송체입니다. NHE1은 N-말단과 C-말단이 모두 세포질을 향하는 12개의 막관통 나선(TMHs)으로 구성됩니다. 주요 수송 세그먼트에는 TM IV, VII, IX가 포함되며, 재진입 루프 IL2 및 IL4와 구조적 안정성을 부여하는 세포외 루프 5 (EL5)의 글리코실화 부위도 있습니다. 확장된 약 315개 잔기의 C-말단 세포질 도메인에는 수많은 세린/트레오닌 인산화 부위와 에즈린 결합 모티프 (잔기 553–564)가 포함되어 NHE1을 액틴 세포골격에 고정시킵니다. NHE1은 세포 내 산성화에 의해 알로스테릭하게 활성화되며, 이는 비수송 조절 부위 (힐 계수 ~3)에 양성자 결합을 촉진하여 박테리아 NhaA 수송체와 유사하게 나선 기울기 및 물로 채워진 접근 경로를 통해 내부 지향 상태에서 외부 지향 상태로의 구조적 변화를 유발합니다. 이는 직접적인 에너지 입력 없이 막관통 Na+ 기울기를 구동력으로 사용하여 세포외 Na+ (Km 5–50 mM)와 세포내 H+를 1:1의 화학량론으로 전기발생적으로 교환합니다. 이를 통해 산증 후 pHi의 빠른 회복, Na+ 유입을 통한 세포 부피 조절, ERM-액틴 상호작용을 통한 라멜리포디움 돌출의 스캐폴딩이 가능합니다. 호르몬 및 성장 인자 신호는 NHERF1, ERK, PKA 경로를 통해 C-말단 세린 (예: S703, Thr653)을 인산화하여 pHi 설정점을 알칼리성 쪽으로 이동시킬 수 있습니다. 병리학적으로, 허혈-재관류 손상 시 산성-활성화된 NHE1은 역방향 NCX 활동을 통해 세포독성 Na+/Ca2+ 과부하를 촉진하여 심근경색을 악화시킵니다. 만성 NHE1 상향 조절은 또한 주변 세포 알칼리화 및 기질 리모델링을 통한 종양 침습 및 심장 비대증과 관련이 있습니다.

사용 정보

응용

WB

희석

WB

1:500

반응성

Mouse, Human, Amphibian, Fish, Avian, Vertebrates

출처

Mouse Monoclonal Antibody

MW

~100-110 kDa

보관 완충액

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

보관
(수령일로부터)

-20°C (avoid freeze-thaw cycles), 2 years

실험 방법
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. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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 1:500), 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 1. 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.

실험 방법

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.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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 1:500), 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

1. 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.

참조

https://pubmed.ncbi.nlm.nih.gov/34108458/
https://pubmed.ncbi.nlm.nih.gov/33273619/

적용 데이터

WB

Selleck 검증

Lane 1: U87MG, Lane 2: THP-1, Lane 3: 22RV1, Lane 4: Mouse brain