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특이성	ApoER2 Antibody [K2M19]는 총 ApoER2 단백질의 내인성 수준을 인식합니다.
배경	lrp8 유전자에 의해 암호화되는 아포지단백질 E 수용체 2(apoER2)는 저밀도 지단백질 수용체(LDLR) 계열에 속하는 제1형 막관통 단백질입니다. ApoER2는 발달 중 피질 층화, 문측 이동 경로(rostral migratory stream)에서의 개재신경원 전구체 이동, 성체 뇌의 학습 및 기억 조절에 핵심적인 역할을 합니다. 다른 LDLR 계열 구성원과 달리 apoER2는 뇌에 더 풍부하며, 특히 뉴런에서 대안적 스플라이싱 사건의 빈도가 높습니다. 구조적으로 apoER2는 모듈식이며, 개별 엑손은 특정 기능적 도메인을 암호화합니다. 이는 LDLR 계열에 전형적인 5개의 기능적 도메인을 포함합니다. 인간 아포지단백질 E(ApoE)는 세 가지 이소형(ApoE2, ApoE3, ApoE4)을 가지며, 이들은 130번 및 176번 위치에 시스테인 및 아르기닌 잔기의 존재 여부에 따라 다릅니다. ApoE2는 두 위치 모두에 시스테인을 가지며, ApoE4는 알츠하이머병(AD)의 유전적 위험 인자로 간주됩니다. 반대로 ApoE2는 신경 보호 효과가 있어 AD 위험을 거의 50% 감소시킵니다. ApoER2는 뇌의 신경 발달 및 시냅스 가소성에 중요하며, 특정 기능적 엑손이 최종 전사체에서 제외되는 캐세트 엑손 스플라이싱 사건이 풍부합니다. 이러한 대안적 스플라이싱 사건은 apoER2 기능에 영향을 미치는데, 각 엑손은 별개의 단백질 기능 도메인을 암호화하는 경향이 있기 때문입니다.

특이성

ApoER2 Antibody [K2M19]는 총 ApoER2 단백질의 내인성 수준을 인식합니다.

배경

lrp8 유전자에 의해 암호화되는 아포지단백질 E 수용체 2(apoER2)는 저밀도 지단백질 수용체(LDLR) 계열에 속하는 제1형 막관통 단백질입니다. ApoER2는 발달 중 피질 층화, 문측 이동 경로(rostral migratory stream)에서의 개재신경원 전구체 이동, 성체 뇌의 학습 및 기억 조절에 핵심적인 역할을 합니다. 다른 LDLR 계열 구성원과 달리 apoER2는 뇌에 더 풍부하며, 특히 뉴런에서 대안적 스플라이싱 사건의 빈도가 높습니다. 구조적으로 apoER2는 모듈식이며, 개별 엑손은 특정 기능적 도메인을 암호화합니다. 이는 LDLR 계열에 전형적인 5개의 기능적 도메인을 포함합니다. 인간 아포지단백질 E(ApoE)는 세 가지 이소형(ApoE2, ApoE3, ApoE4)을 가지며, 이들은 130번 및 176번 위치에 시스테인 및 아르기닌 잔기의 존재 여부에 따라 다릅니다. ApoE2는 두 위치 모두에 시스테인을 가지며, ApoE4는 알츠하이머병(AD)의 유전적 위험 인자로 간주됩니다. 반대로 ApoE2는 신경 보호 효과가 있어 AD 위험을 거의 50% 감소시킵니다. ApoER2는 뇌의 신경 발달 및 시냅스 가소성에 중요하며, 특정 기능적 엑손이 최종 전사체에서 제외되는 캐세트 엑손 스플라이싱 사건이 풍부합니다. 이러한 대안적 스플라이싱 사건은 apoER2 기능에 영향을 미치는데, 각 엑손은 별개의 단백질 기능 도메인을 암호화하는 경향이 있기 때문입니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000-1:10000	1:10 - 1:100

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

106 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: 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 968. 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: 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

968. 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/32848602/

적용 데이터

WB

Selleck 검증

Lane 1: Mouse brain
Lane 2: C6
Lane 3: Neuro-2a