IP3 Receptor 1 Antibody [M15D2]

적용: 반응성:Human, Mouse, Rat

Lane 1: Mouse brain

고객 리뷰

실험 필수품

WB
Recommended SDS-PAGE separating gel concentration: 5%.
Recommended wet transfer conditions: 250 mA, 180 min.

사용 정보

희석
WB1:1000 IP1:50

응용
WB, IP

반응성
Human, Mouse, Rat

출처
Rabbit Monoclonal Antibody

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

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

예측 분자량
320 kDa

양성 대조군	Mouse brain; Rat brain
음성 대조군

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.

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.

Datasheet & SDS

생물학적 설명

특이성
IP3 Receptor 1 Antibody [M15D2] recognizes endogenous levels of total IP3R1 protein. This antibody may also detect IP3R2 and IP3R3 as the antigen is highly conserved.

세포 내 위치
Cytoplasm, Cytoplasmic vesicle, Endoplasmic reticulum, Membrane

Uniprot ID
Q14643

클론
M15D2

동의어
IP3 receptor,IP3 Receptor 1

배경
Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are large, ubiquitous ion channels that mediate Ca²⁺ release from intracellular stores like the endoplasmic reticulum (ER) and Golgi apparatus. Vertebrates have three closely related InsP3R subtypes (IP3R1-3) that form homo- and heterotetrameric channels. Each InsP3R subunit is composed of approximately 2700 amino acids and contains ten domains responsible for channel stability, ion transport, ligand binding, and regulation. Activation of InsP3Rs requires binding of inositol 1,4,5-trisphosphate (IP3) and Ca²⁺, leading to the release of Ca²⁺ into the cytosol, which is crucial for processes like muscle contraction, secretion, proliferation, and apoptosis. InsP3R1, the most studied subtype, is predominantly expressed in cerebellar Purkinje cells.The type I inositol-1,4,5-trisphosphate receptor (InsP3R1) is the most widely expressed and extensively studied member of the InsP3R family. Beyond its activation by InsP3 and Ca²⁺, InsP3R1 is regulated through several mechanisms, including phosphorylation by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). These kinases phosphorylate the receptor at two specific serine residues, S1588 and S1755, in the mouse InsP3R1, further modulating its activity.

배경

Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are large, ubiquitous ion channels that mediate Ca²⁺ release from intracellular stores like the endoplasmic reticulum (ER) and Golgi apparatus. Vertebrates have three closely related InsP3R subtypes (IP3R1-3) that form homo- and heterotetrameric channels. Each InsP3R subunit is composed of approximately 2700 amino acids and contains ten domains responsible for channel stability, ion transport, ligand binding, and regulation. Activation of InsP3Rs requires binding of inositol 1,4,5-trisphosphate (IP3) and Ca²⁺, leading to the release of Ca²⁺ into the cytosol, which is crucial for processes like muscle contraction, secretion, proliferation, and apoptosis. InsP3R1, the most studied subtype, is predominantly expressed in cerebellar Purkinje cells.The type I inositol-1,4,5-trisphosphate receptor (InsP3R1) is the most widely expressed and extensively studied member of the InsP3R family. Beyond its activation by InsP3 and Ca²⁺, InsP3R1 is regulated through several mechanisms, including phosphorylation by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). These kinases phosphorylate the receptor at two specific serine residues, S1588 and S1755, in the mouse InsP3R1, further modulating its activity.

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

단백질 크기	PVDF 전이막 기공 크기
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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단백질 크기	분리 젤 퍼센트
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%