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특이성	Phospho-NMDAR2B (Ser1303) Antibody [G14F14]는 S1303에서 인산화된 경우에만 내인성 NMDAR2B 단백질 수준을 인식합니다.
배경	N-메틸-D-아스파르테이트 수용체 (NMDAR)는 적어도 하나의 NR1 서브유닛과 하나의 NR2A-D 서브유닛으로 구성된 헤테로다이머입니다. NR1 전사체의 대체 스플라이싱과 NR2 서브유닛의 차등 발현을 통해 기능적 특성 및 뇌 분포가 다른 다양한 수용체 이소폼이 생성됩니다. NR1 서브유닛은 공동 작용제로 글리신을 결합하고, NR2 서브유닛은 주요 신경전달물질인 글루타메이트를 결합합니다. NMDAR의 활성화는 이온 채널을 열어 나트륨(Na⁺) 및 칼슘(Ca²⁺) 이온이 세포 내로 유입되고 칼륨(K⁺) 이온이 세포 밖으로 유출되도록 합니다. 단백질 키나아제 C(PKC)는 NR1 서브유닛을 Ser890 및 Ser896에서 인산화하여 칼모듈린에 대한 친화도를 감소시키고, 이로 인해 칼모듈린의 수용체 음성 조절을 억제합니다. 단백질 키나아제 A(PKA)는 NR1을 Ser897에서 인산화하며, 이는 칼시뉴린 매개 수용체 억제를 상쇄하는 것으로 알려져 있습니다.NMDAR은 학습, 신경 발달 및 신경 가소성에 필수적인 장기 강화 및 느린 시냅스 후 흥분과 같은 시냅스 과정에서 중요한 역할을 합니다. NR2B 서브유닛의 Ser1303 인산화는 신경 흥분 독성 동안 칼슘 과부하를 유발하는 것과 관련이 있으며, 이 부위의 탈인산화는 신경 보호 효과를 제공할 수 있습니다.

특이성

Phospho-NMDAR2B (Ser1303) Antibody [G14F14]는 S1303에서 인산화된 경우에만 내인성 NMDAR2B 단백질 수준을 인식합니다.

배경

N-메틸-D-아스파르테이트 수용체 (NMDAR)는 적어도 하나의 NR1 서브유닛과 하나의 NR2A-D 서브유닛으로 구성된 헤테로다이머입니다. NR1 전사체의 대체 스플라이싱과 NR2 서브유닛의 차등 발현을 통해 기능적 특성 및 뇌 분포가 다른 다양한 수용체 이소폼이 생성됩니다. NR1 서브유닛은 공동 작용제로 글리신을 결합하고, NR2 서브유닛은 주요 신경전달물질인 글루타메이트를 결합합니다. NMDAR의 활성화는 이온 채널을 열어 나트륨(Na⁺) 및 칼슘(Ca²⁺) 이온이 세포 내로 유입되고 칼륨(K⁺) 이온이 세포 밖으로 유출되도록 합니다. 단백질 키나아제 C(PKC)는 NR1 서브유닛을 Ser890 및 Ser896에서 인산화하여 칼모듈린에 대한 친화도를 감소시키고, 이로 인해 칼모듈린의 수용체 음성 조절을 억제합니다. 단백질 키나아제 A(PKA)는 NR1을 Ser897에서 인산화하며, 이는 칼시뉴린 매개 수용체 억제를 상쇄하는 것으로 알려져 있습니다.NMDAR은 학습, 신경 발달 및 신경 가소성에 필수적인 장기 강화 및 느린 시냅스 후 흥분과 같은 시냅스 과정에서 중요한 역할을 합니다. NR2B 서브유닛의 Ser1303 인산화는 신경 흥분 독성 동안 칼슘 과부하를 유발하는 것과 관련이 있으며, 이 부위의 탈인산화는 신경 보호 효과를 제공할 수 있습니다.

사용 정보

응용

WB

희석

WB

1:1000-1:5000

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

180 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, Phosphatase 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, Phosphatase 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, Phosphatase 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: 5%. 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 ( recommending 5% BSA 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 1250. 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, Phosphatase 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, Phosphatase 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, Phosphatase 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: 5%. 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 ( recommending 5% BSA 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

1250. 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/15470155/
https://pubmed.ncbi.nlm.nih.gov/22479519/

적용 데이터

WB

Selleck 검증

Lane 1: Rat brain
Lane 2: Rat brain (alkaline phosphatase treated)
Lane 3: Human brain
Lane 4: Human brain (alkaline phosphatase treated)