HACE1 Antibody [A17B18]

적용: 반응성:Mouse, Rat, Human

Lane 1: HEK293, Lane 2: SH-SY5Y, Lane 3: Mouse brain, Lane 4: Rat brain

사용 정보

희석
WB1:1000 - 1:10000

응용
WB

반응성
Mouse, Rat, 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
102 kDa 102 kDa
^*예측 분자량과 실제 분자량이 다른 이유는 무엇입니까? 다음과 같은 이유로 예측 분자량과 실제 단백질 분자량 간에 차이가 있을 수 있습니다.

양성 대조군	Rat brain; Mouse brain; Human fetal brain; HEK293; SH-SY5Y; 293T
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. 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/NP-40 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/NP-40 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: 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: 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/NP-40 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/NP-40 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/NP-40 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: 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: 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
HACE1 Antibody [A17B18] recognizes endogenous levels of total HACE1 protein.

Uniprot ID
Q8IYU2

클론
A17B18

동의어
KIAA1320, HACE1, E3 ubiquitin-protein ligase HACE1, HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1, HECT-type E3 ubiquitin transferase HACE1

배경
The HACE1 (HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1) gene is located on chromosome 6 and encodes a protein composed of 909 amino acids. HACE1 is strongly expressed in various human tissues, including the heart, brain, placenta, pancreas, and both fetal and adult kidneys. HACE1 mRNA is ubiquitously expressed across normal human tissues. Reduced expression levels of HACE1 are strongly associated with hypermethylation of two CpG islands located upstream of its gene locus, suggesting epigenetic regulation plays a role in its silencing. HACE1 is frequently downregulated in several types of cancer, including natural killer/T-cell lymphoma (NKTCL), colorectal cancer, and gastric cancer. Subcellularly, HACE1 localizes mainly to the endoplasmic reticulum and cytosol, although only low levels of endogenous protein are typically detected. Functionally, HACE1 acts as an E3 ubiquitin ligase and partners with the E2 enzyme UBCH7 to catalyze ubiquitination of target proteins. It is notably involved in phosphorylation-dependent degradation of cyclin D1, thereby playing a role in inhibiting cell cycle progression. HACE1 also preferentially binds to the active, GTP-bound form of the small GTPase Rac1 and promotes its polyubiquitination. This activity is essential for Rac1 degradation in response to cytotoxic necrotizing factor 1 (CNF1), facilitating bacterial invasion of endothelial cell monolayers, and highlighting a role for HACE1 in innate immune defense mechanisms. loss of HACE1 leads to the spontaneous development of late-onset tumors, further supporting its function as a tumor suppressor. The gene lies within the 6q21 chromosomal region which is a hotspot implicated in multiple human cancers, underscoring its clinical significance in tumorigenesis.

배경

The HACE1 (HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1) gene is located on chromosome 6 and encodes a protein composed of 909 amino acids. HACE1 is strongly expressed in various human tissues, including the heart, brain, placenta, pancreas, and both fetal and adult kidneys. HACE1 mRNA is ubiquitously expressed across normal human tissues. Reduced expression levels of HACE1 are strongly associated with hypermethylation of two CpG islands located upstream of its gene locus, suggesting epigenetic regulation plays a role in its silencing. HACE1 is frequently downregulated in several types of cancer, including natural killer/T-cell lymphoma (NKTCL), colorectal cancer, and gastric cancer. Subcellularly, HACE1 localizes mainly to the endoplasmic reticulum and cytosol, although only low levels of endogenous protein are typically detected. Functionally, HACE1 acts as an E3 ubiquitin ligase and partners with the E2 enzyme UBCH7 to catalyze ubiquitination of target proteins. It is notably involved in phosphorylation-dependent degradation of cyclin D1, thereby playing a role in inhibiting cell cycle progression. HACE1 also preferentially binds to the active, GTP-bound form of the small GTPase Rac1 and promotes its polyubiquitination. This activity is essential for Rac1 degradation in response to cytotoxic necrotizing factor 1 (CNF1), facilitating bacterial invasion of endothelial cell monolayers, and highlighting a role for HACE1 in innate immune defense mechanisms. loss of HACE1 leads to the spontaneous development of late-onset tumors, further supporting its function as a tumor suppressor. The gene lies within the 6q21 chromosomal region which is a hotspot implicated in multiple human cancers, underscoring its clinical significance in tumorigenesis.

참고문헌
https://pubmed.ncbi.nlm.nih.gov/34815381/ https://pubmed.ncbi.nlm.nih.gov/38364733/

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%