PAN TEAD Antibody [A12P9]

적용: 반응성:Human

Lane 1: HeLa, Lane 2: SW480

사용 정보

희석
WB1:1000

응용
WB

반응성
Human

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 관찰 분자량
48 kDa 54 kDa
^*예측 분자량과 실제 분자량이 다른 이유는 무엇입니까? 다음과 같은 이유로 예측 분자량과 실제 단백질 분자량 간에 차이가 있을 수 있습니다.

양성 대조군	HeLa; SW480
음성 대조군

실험 방법

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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 1. 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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

1. 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
PAN TEAD Antibody [A12P9] detects endogenous levels of total Pan-TEAD protein.

세포 내 위치
Nucleus

Uniprot ID
P28347; Q15562; Q99594; Q15561

클론
A12P9

동의어
RTEF1, TCF13L1, TEF3, TEAD4, Transcriptional enhancer factor TEF-3, TEA domain family member 4, Transcription factor 13-like 1, Transcription factor RTEF-1, TEAD-4

배경
The TEAD family of transcription factors, comprising TEAD1, TEAD2, TEAD3, and TEAD4, is a conserved group of DNA-binding proteins that regulate genes controlling cell proliferation, differentiation, survival, apoptosis, tissue growth, and regeneration. They act as central downstream effectors of the Hippo signaling pathway, integrating mechanical and biochemical inputs to maintain tissue homeostasis and control organ size. All TEAD proteins share an N-terminal TEA DNA-binding domain (~70–75 amino acids) with a helix-turn-helix fold that recognizes MCAT elements in target promoters, and a C-terminal YAP-binding domain (YBD) with an immunoglobulin-like fold that recruits the transcriptional coactivators YAP and TAZ. These coactivators are essential for robust transcriptional activation of target genes such as CTGF and CYR61, since TEADs alone have low intrinsic activity. Coordinated TEAD function influences cytoskeletal dynamics, wound healing, and tissue regeneration. Dysregulation of any TEAD family member, often through aberrant YAP/TAZ activation, drives oncogenic transcriptional programs, promotes metastasis, and is associated with developmental disorders, fibroproliferative diseases, and cardiomyopathies.

배경

The TEAD family of transcription factors, comprising TEAD1, TEAD2, TEAD3, and TEAD4, is a conserved group of DNA-binding proteins that regulate genes controlling cell proliferation, differentiation, survival, apoptosis, tissue growth, and regeneration. They act as central downstream effectors of the Hippo signaling pathway, integrating mechanical and biochemical inputs to maintain tissue homeostasis and control organ size. All TEAD proteins share an N-terminal TEA DNA-binding domain (~70–75 amino acids) with a helix-turn-helix fold that recognizes MCAT elements in target promoters, and a C-terminal YAP-binding domain (YBD) with an immunoglobulin-like fold that recruits the transcriptional coactivators YAP and TAZ. These coactivators are essential for robust transcriptional activation of target genes such as CTGF and CYR61, since TEADs alone have low intrinsic activity. Coordinated TEAD function influences cytoskeletal dynamics, wound healing, and tissue regeneration. Dysregulation of any TEAD family member, often through aberrant YAP/TAZ activation, drives oncogenic transcriptional programs, promotes metastasis, and is associated with developmental disorders, fibroproliferative diseases, and cardiomyopathies.

참고문헌
https://pubmed.ncbi.nlm.nih.gov/18579750/ https://pubmed.ncbi.nlm.nih.gov/20123908/

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%