데이터시트

생물학적 설명

특이성	IP3 Receptor 1 Antibody [M15D2]는 총 IP3R1 단백질의 내인성 수준을 인식합니다. 이 항체는 항원이 고도로 보존되어 있으므로 IP3R2 및 IP3R3도 검출할 수 있습니다.
배경	이노시톨-1,4,5-트리포스페이트 수용체(InsP3Rs)는 소포체(ER) 및 골지체와 같은 세포내 저장고로부터 Ca²⁺ 방출을 매개하는 크고 보편적인 이온 채널입니다. 척추동물은 호모 및 헤테로테트라머 채널을 형성하는 세 가지 밀접하게 관련된 InsP3R 서브타입(IP3R1-3)을 가지고 있습니다. 각 InsP3R 서브유닛은 약 2700개의 아미노산으로 구성되며 채널 안정성, 이온 수송, 리간드 결합 및 조절을 담당하는 10개의 도메인을 포함합니다. InsP3Rs 활성화는 이노시톨 1,4,5-트리포스페이트(IP3) 및 Ca²⁺의 결합을 필요로 하며, 이는 세포질로의 Ca²⁺ 방출로 이어지고, 이는 근육 수축, 분비, 증식 및 세포자멸사와 같은 과정에 중요합니다. 가장 많이 연구된 서브타입인 InsP3R1은 주로 소뇌 푸르키녜 세포에서 발현됩니다. 유형 I 이노시톨-1,4,5-트리포스페이트 수용체(InsP3R1)는 InsP3R 계열에서 가장 널리 발현되고 광범위하게 연구된 구성원입니다. InsP3 및 Ca²⁺에 의한 활성화 외에도 InsP3R1은 cAMP 의존성 단백질 키나제(PKA) 및 cGMP 의존성 단백질 키나제(PKG)에 의한 인산화를 포함한 여러 메커니즘을 통해 조절됩니다. 이 키나제는 마우스 InsP3R1의 S1588 및 S1755라는 두 가지 특정 세린 잔기에서 수용체를 인산화하여 활동을 더욱 조절합니다.

특이성

IP3 Receptor 1 Antibody [M15D2]는 총 IP3R1 단백질의 내인성 수준을 인식합니다. 이 항체는 항원이 고도로 보존되어 있으므로 IP3R2 및 IP3R3도 검출할 수 있습니다.

배경

이노시톨-1,4,5-트리포스페이트 수용체(InsP3Rs)는 소포체(ER) 및 골지체와 같은 세포내 저장고로부터 Ca²⁺ 방출을 매개하는 크고 보편적인 이온 채널입니다. 척추동물은 호모 및 헤테로테트라머 채널을 형성하는 세 가지 밀접하게 관련된 InsP3R 서브타입(IP3R1-3)을 가지고 있습니다. 각 InsP3R 서브유닛은 약 2700개의 아미노산으로 구성되며 채널 안정성, 이온 수송, 리간드 결합 및 조절을 담당하는 10개의 도메인을 포함합니다. InsP3Rs 활성화는 이노시톨 1,4,5-트리포스페이트(IP3) 및 Ca²⁺의 결합을 필요로 하며, 이는 세포질로의 Ca²⁺ 방출로 이어지고, 이는 근육 수축, 분비, 증식 및 세포자멸사와 같은 과정에 중요합니다. 가장 많이 연구된 서브타입인 InsP3R1은 주로 소뇌 푸르키녜 세포에서 발현됩니다. 유형 I 이노시톨-1,4,5-트리포스페이트 수용체(InsP3R1)는 InsP3R 계열에서 가장 널리 발현되고 광범위하게 연구된 구성원입니다. InsP3 및 Ca²⁺에 의한 활성화 외에도 InsP3R1은 cAMP 의존성 단백질 키나제(PKA) 및 cGMP 의존성 단백질 키나제(PKG)에 의한 인산화를 포함한 여러 메커니즘을 통해 조절됩니다. 이 키나제는 마우스 InsP3R1의 S1588 및 S1755라는 두 가지 특정 세린 잔기에서 수용체를 인산화하여 활동을 더욱 조절합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

320 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 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: 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: 250 mA, 180 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 796. 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 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: 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: 250 mA, 180 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

796. 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/26458101/
https://pubmed.ncbi.nlm.nih.gov/30745293/

적용 데이터

WB

Selleck 검증

Lane 1: Mouse brain