Datasheet

Biological Description

Specificity	TRPC1 Rabbit mAb recognizes endogenous levels of total TRPC1 protein.
Background	TRP channels are a large family of proteins found throughout the body. Structurally, they consist of four subunits, each with six transmembrane domains, resembling voltage-dependent channels but lacking positive charges and typically being voltage-insensitive. The TRP family is categorized into six subfamilies, with the most notable being the canonical TRP (TRPC1–7), melastatin-related TRP (TRPM1–8), and vanilloid receptor-related TRP (TRPV1–6) subfamilies. Certain TRP channels play a role in cell proliferation and cancer progression. TRPC1, in particular, has been implicated in enhancing cell proliferation and regulating cell migration, both of which contribute to cancer aggressiveness. As a non-selective cation channel (PCa/PNa ∼ 1), TRPC1 participates in store-operated calcium entry (also known as capacitative calcium entry) alongside Orai1, with activation mediated by STIM1, the sensor of endoplasmic reticulum calcium levels. TRPC1 is essential for directed cell migration in response to chemotactic signals. High extracellular calcium ([Ca2+]o) levels stimulate the calcium-sensing receptor, leading to increased cell proliferation and TRPC1 expression. Additionally, TRPC1 facilitates ERK1/2 phosphorylation following calcium-sensing receptor activation. Knockdown of TRPC1 can halt cell growth by arresting the cell cycle at the G0/G1 phase in endothelial progenitor cells or causing incomplete cytokinesis in gliomas. Furthermore, TRPC1 is involved in epidermal growth factor (EGF)-induced cell migration.

Specificity

TRPC1 Rabbit mAb recognizes endogenous levels of total TRPC1 protein.

Background

TRP channels are a large family of proteins found throughout the body. Structurally, they consist of four subunits, each with six transmembrane domains, resembling voltage-dependent channels but lacking positive charges and typically being voltage-insensitive. The TRP family is categorized into six subfamilies, with the most notable being the canonical TRP (TRPC1–7), melastatin-related TRP (TRPM1–8), and vanilloid receptor-related TRP (TRPV1–6) subfamilies. Certain TRP channels play a role in cell proliferation and cancer progression. TRPC1, in particular, has been implicated in enhancing cell proliferation and regulating cell migration, both of which contribute to cancer aggressiveness. As a non-selective cation channel (PCa/PNa ∼ 1), TRPC1 participates in store-operated calcium entry (also known as capacitative calcium entry) alongside Orai1, with activation mediated by STIM1, the sensor of endoplasmic reticulum calcium levels. TRPC1 is essential for directed cell migration in response to chemotactic signals. High extracellular calcium ([Ca2+]o) levels stimulate the calcium-sensing receptor, leading to increased cell proliferation and TRPC1 expression. Additionally, TRPC1 facilitates ERK1/2 phosphorylation following calcium-sensing receptor activation. Knockdown of TRPC1 can halt cell growth by arresting the cell cycle at the G0/G1 phase in endothelial progenitor cells or causing incomplete cytokinesis in gliomas. Furthermore, TRPC1 is involved in epidermal growth factor (EGF)-induced cell migration.

Usage Information

Application

WB

Dilution

WB

1:1000-1:10000

Reactivity

Human, Mouse, Rat

Source

Rabbit

MW

91 kDa

Storage Buffer

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

Storage
(from the date of receipt)

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

Exprimental Methods
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.

Exprimental Methods

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.

References

[1] Tajeddine N, et al. J Biol Chem. 2012 May 11;287(20):16146-57.

Application Data

WB

Validated by Selleck

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