데이터시트

생물학적 설명

특이성	TRPC1 Antibody [J16G9]는 총 TRPC1 단백질의 내인성 수준을 인식합니다.
배경	TRP Channel은 몸 전체에서 발견되는 큰 단백질 패밀리입니다. 구조적으로, 이들은 각각 6개의 막관통 도메인을 가진 4개의 소단위로 구성되며, 전압 의존성 채널과 유사하지만 양전하가 없고 일반적으로 전압에 둔감합니다. TRP 패밀리는 6개의 하위 패밀리로 분류되며, 가장 주목할 만한 것은 정식 TRP (TRPC17), 멜라스타틴 관련 TRP (TRPM18) 및 바닐로이드 수용체 관련 TRP (TRPV16) 하위 패밀리입니다. 특정 TRP Channel은 세포 증식 및 암 진행에 중요한 역할을 합니다. 특히 TRPC1은 세포 증식 증진 및 세포 이동 조절에 관여하는 것으로 알려져 있으며, 이 두 가지 모두 암의 공격성에 기여합니다. 비선택적 양이온 채널(PCa/PNa ∼ 1)로서 TRPC1은 Orai1과 함께 저장 작동성 칼슘 유입(capacitative calcium entry라고도 함)에 참여하며, 소포체 칼슘 수준의 센서인 STIM1에 의해 활성화됩니다. TRPC1은 화학 주성 신호에 반응하는 지향성 세포 이동에 필수적입니다. 높은 세포외 칼슘([Ca2+]o) 수준은 칼슘 감지 수용체를 자극하여 세포 증식 및 TRPC1 발현을 증가시킵니다. 또한 TRPC1은 칼슘 감지 수용체 활성화 후 ERK1/2 인산화를 촉진합니다. TRPC1의 녹다운은 내피 전구 세포에서 G0/G1기에서 세포 주기를 정지시키거나 신경교종에서 불완전한 세포 분열을 유발하여 세포 성장을 멈출 수 있습니다. 또한 TRPC1은 상피 성장 인자(EGF) 유도 세포 이동에 관여합니다.

특이성

TRPC1 Antibody [J16G9]는 총 TRPC1 단백질의 내인성 수준을 인식합니다.

배경

TRP Channel은 몸 전체에서 발견되는 큰 단백질 패밀리입니다. 구조적으로, 이들은 각각 6개의 막관통 도메인을 가진 4개의 소단위로 구성되며, 전압 의존성 채널과 유사하지만 양전하가 없고 일반적으로 전압에 둔감합니다. TRP 패밀리는 6개의 하위 패밀리로 분류되며, 가장 주목할 만한 것은 정식 TRP (TRPC17), 멜라스타틴 관련 TRP (TRPM18) 및 바닐로이드 수용체 관련 TRP (TRPV16) 하위 패밀리입니다. 특정 TRP Channel은 세포 증식 및 암 진행에 중요한 역할을 합니다. 특히 TRPC1은 세포 증식 증진 및 세포 이동 조절에 관여하는 것으로 알려져 있으며, 이 두 가지 모두 암의 공격성에 기여합니다. 비선택적 양이온 채널(PCa/PNa ∼ 1)로서 TRPC1은 Orai1과 함께 저장 작동성 칼슘 유입(capacitative calcium entry라고도 함)에 참여하며, 소포체 칼슘 수준의 센서인 STIM1에 의해 활성화됩니다. TRPC1은 화학 주성 신호에 반응하는 지향성 세포 이동에 필수적입니다. 높은 세포외 칼슘([Ca2+]o) 수준은 칼슘 감지 수용체를 자극하여 세포 증식 및 TRPC1 발현을 증가시킵니다. 또한 TRPC1은 칼슘 감지 수용체 활성화 후 ERK1/2 인산화를 촉진합니다. TRPC1의 녹다운은 내피 전구 세포에서 G0/G1기에서 세포 주기를 정지시키거나 신경교종에서 불완전한 세포 분열을 유발하여 세포 성장을 멈출 수 있습니다. 또한 TRPC1은 상피 성장 인자(EGF) 유도 세포 이동에 관여합니다.

사용 정보

응용

WB

희석

WB

1:1000-1:10000

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

91 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: 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 1311. 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: 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

1311. 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/22451676/

적용 데이터

WB

Selleck 검증

Lane 1: SH-SY5Y
Lane 2: HepG2
Lane 3: Mouse brain
Lane 4: Rat brain