데이터시트

생물학적 설명

특이성	SEP15 Antibody [A2N16]는 총 SEP15 단백질의 내인성 수준을 인식합니다.
배경	Sep15 (15-kDa 셀레노프로테인)는 티오레독신 유사 특성을 가진 소포체(ER) 국소화 단백질로, Glycoprotein 품질 관리에 관여합니다. 이는 Glycoprotein 접힘의 핵심 요소인 UDP-glucose:Glycoprotein 글루코실전달효소와 상호작용하여 기능합니다. Sep15의 정확한 생물학적 역할은 여전히 불분명하지만, 그 발현은 식이 셀레늄과 미접힘 단백질 반응 모두에 의해 영향을 받는 것으로 알려져 있습니다. Sep15는 적응성 ER 스트레스 조건에서 상향 조절되며, 이는 세포 스트레스 반응에서 역할을 한다는 것을 시사합니다. 인간 Sep15 유전자는 염색체 1p31에 위치하며 — 이 유전체 영역은 다양한 암에서 자주 결실되거나 돌연변이됩니다. 또한, Sep15 유전자의 두 가지 다형성 부위는 셀레늄 섭취에 대한 발현 반응 방식에 영향을 미칩니다. 특히, Sep15 발현은 여러 암 유형에서 감소합니다. 전립선암 세포주 및 간세포암종 조직에서, 그리고 악성 폐암, 유방암, 전립선암 및 간암 종양에서 비암성 대응 기관에 비해 낮은 수준이 관찰되었습니다. 이러한 발견은 Sep15가 암 진행과 관련된 Glycoproteins의 접힘 및/또는 분비를 조절함으로써 종양 억제제 역할을 할 수 있음을 시사합니다.

특이성

SEP15 Antibody [A2N16]는 총 SEP15 단백질의 내인성 수준을 인식합니다.

배경

Sep15 (15-kDa 셀레노프로테인)는 티오레독신 유사 특성을 가진 소포체(ER) 국소화 단백질로, Glycoprotein 품질 관리에 관여합니다. 이는 Glycoprotein 접힘의 핵심 요소인 UDP-glucose:Glycoprotein 글루코실전달효소와 상호작용하여 기능합니다. Sep15의 정확한 생물학적 역할은 여전히 불분명하지만, 그 발현은 식이 셀레늄과 미접힘 단백질 반응 모두에 의해 영향을 받는 것으로 알려져 있습니다. Sep15는 적응성 ER 스트레스 조건에서 상향 조절되며, 이는 세포 스트레스 반응에서 역할을 한다는 것을 시사합니다. 인간 Sep15 유전자는 염색체 1p31에 위치하며 — 이 유전체 영역은 다양한 암에서 자주 결실되거나 돌연변이됩니다. 또한, Sep15 유전자의 두 가지 다형성 부위는 셀레늄 섭취에 대한 발현 반응 방식에 영향을 미칩니다. 특히, Sep15 발현은 여러 암 유형에서 감소합니다. 전립선암 세포주 및 간세포암종 조직에서, 그리고 악성 폐암, 유방암, 전립선암 및 간암 종양에서 비암성 대응 기관에 비해 낮은 수준이 관찰되었습니다. 이러한 발견은 Sep15가 암 진행과 관련된 Glycoproteins의 접힘 및/또는 분비를 조절함으로써 종양 억제제 역할을 할 수 있음을 시사합니다.

사용 정보

응용

WB

희석

WB

1:1000 - 1:10000

반응성

Rat, Human

출처

Rabbit Monoclonal Antibody

MW

18 kDa

보관 완충액

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

보관
(수령일로부터)

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

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: 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. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 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.

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: 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. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 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.

참조

https://pubmed.ncbi.nlm.nih.gov/21768092/

적용 데이터

WB

Selleck 검증

Lane 1: Rat liver, Lane 2: Fetal liver, Lane 3: LnCaP