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Biologische Beschrijving

Specificiteit	TRPC1 Antibody [J16G9] herkent endogene niveaus van totaal TRPC1-eiwit.
Achtergrond	TRP Channel zijn een grote familie van eiwitten die overal in het lichaam worden aangetroffen. Structureel bestaan ze uit vier subeenheden, elk met zes transmembraandomeinen, die lijken op spanningsafhankelijke kanalen, maar zonder positieve ladingen en doorgaans spanningsongevoelig zijn. De TRP-familie is onderverdeeld in zes subfamilies, waarvan de meest opvallende de canonieke TRP (TRPC17), melastatine-gerelateerde TRP (TRPM18) en vanilloïde receptor-gerelateerde TRP (TRPV16) subfamilies zijn. Bepaalde TRP Channel spelen een rol bij celproliferatie en kankerprogressie. TRPC1 is in het bijzonder betrokken bij het verbeteren van celproliferatie en het reguleren van celmigratie, die beide bijdragen aan de agressiviteit van kanker. Als een niet-selectief kationkanaal (PCa/PNa ∼ 1) neemt TRPC1 deel aan store-operated calcium entry (ook bekend als capacitatieve calcium entry) samen met Orai1, waarbij activering wordt gemedieerd door STIM1, de sensor van calciumspiegels in het endoplasmatisch reticulum. TRPC1 is essentieel voor gerichte celmigratie als reactie op chemotactische signalen. Hoge extracellulaire calcium ([Ca2+]o) niveaus stimuleren de calcium-sensing receptor, wat leidt tot verhoogde celproliferatie en TRPC1-expressie. Bovendien faciliteert TRPC1 ERK1/2-fosforylatie na activering van de calcium-sensing receptor. Knockdown van TRPC1 kan celgroei stoppen door de celcyclus in de G0/G1-fase te arresteren in endotheelprogenitorcellen of door onvolledige cytokinese te veroorzaken in gliomen. Bovendien is TRPC1 betrokken bij epidermale groeifactor (EGF)-geïnduceerde celmigratie.

Specificiteit

TRPC1 Antibody [J16G9] herkent endogene niveaus van totaal TRPC1-eiwit.

Achtergrond

TRP Channel zijn een grote familie van eiwitten die overal in het lichaam worden aangetroffen. Structureel bestaan ze uit vier subeenheden, elk met zes transmembraandomeinen, die lijken op spanningsafhankelijke kanalen, maar zonder positieve ladingen en doorgaans spanningsongevoelig zijn. De TRP-familie is onderverdeeld in zes subfamilies, waarvan de meest opvallende de canonieke TRP (TRPC17), melastatine-gerelateerde TRP (TRPM18) en vanilloïde receptor-gerelateerde TRP (TRPV16) subfamilies zijn. Bepaalde TRP Channel spelen een rol bij celproliferatie en kankerprogressie. TRPC1 is in het bijzonder betrokken bij het verbeteren van celproliferatie en het reguleren van celmigratie, die beide bijdragen aan de agressiviteit van kanker. Als een niet-selectief kationkanaal (PCa/PNa ∼ 1) neemt TRPC1 deel aan store-operated calcium entry (ook bekend als capacitatieve calcium entry) samen met Orai1, waarbij activering wordt gemedieerd door STIM1, de sensor van calciumspiegels in het endoplasmatisch reticulum. TRPC1 is essentieel voor gerichte celmigratie als reactie op chemotactische signalen. Hoge extracellulaire calcium ([Ca2+]o) niveaus stimuleren de calcium-sensing receptor, wat leidt tot verhoogde celproliferatie en TRPC1-expressie. Bovendien faciliteert TRPC1 ERK1/2-fosforylatie na activering van de calcium-sensing receptor. Knockdown van TRPC1 kan celgroei stoppen door de celcyclus in de G0/G1-fase te arresteren in endotheelprogenitorcellen of door onvolledige cytokinese te veroorzaken in gliomen. Bovendien is TRPC1 betrokken bij epidermale groeifactor (EGF)-geïnduceerde celmigratie.

Gebruiksinformatie

Toepassing

WB

Verdunning

WB

1:1000-1:10000

Reactiviteit

Human, Mouse, Rat

Bron

Rabbit Monoclonal Antibody

MW

91 kDa

Opslagbuffer

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

Opslag
(Vanaf de datum van ontvangst)

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

Experimentele Methoden
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.

Experimentele Methoden

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.

Referenties

https://pubmed.ncbi.nlm.nih.gov/22451676/

Toepassingsgegevens

WB

Gevalideerd door Selleck

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