Datasheet

Biologische Beschrijving

Specificiteit	UBE3A Antibody [P7K8] herkent endogene niveaus van totaal UBE3A-eiwit.
Achtergrond	UBE3A, ook bekend als E6AP (E6 Associated Protein), is een E3 ubiquitine ligase en het eerst geïdentificeerde lid van de HECT (Homologous to the E6 Carboxyl Terminus) familie van E3 ligases. Het wordt gekenmerkt door een sterk geconserveerd C-terminaal katalytisch domein bestaande uit 350 residuen. Veranderingen in de E6AP-functie, hetzij door activering of verlies, zijn gekoppeld aan verschillende menselijke ziekten. Verlies van E6AP-functie als gevolg van deletie, imprintingdefecten of mutaties in het UBE3A-gen gelegen in de 15q11–13 chromosoomregio wordt geassocieerd met Angelman-syndroom, een neurologische aandoening. De meeste natuurlijke mutaties in UBE3A leiden tot deleties die verkorte vormen van E6AP creëren waarbij het complete HECT-domein ontbreekt. Ongeveer 10% van de genetische veranderingen zijn echter puntmutaties binnen het E6AP-coderende gebied. Omgekeerd is duplicatie van het UBE3A-gen gesuggereerd als een bijdragende factor in sommige gevallen van autismespectrumstoornis. Het oncogene E6-eiwit van hoogrisico humane papillomavirussen (HPV16 en HPV18) kan UBE3A kapen, wat leidt tot onjuiste ubiquitinatie van specifieke cellulaire eiwitten, met name p53, wat significant is in de context van kankerontwikkeling. In neuronen resulteert verstoring van de UBE3A-functie in verhoogde Arc-expressie en een vermindering van AMPA-receptoren (AMPARs) bij excitatorische synapsen, wat potentieel bijdraagt aan de neurologische symptomen van Angelman-syndroom.

Specificiteit

UBE3A Antibody [P7K8] herkent endogene niveaus van totaal UBE3A-eiwit.

Achtergrond

UBE3A, ook bekend als E6AP (E6 Associated Protein), is een E3 ubiquitine ligase en het eerst geïdentificeerde lid van de HECT (Homologous to the E6 Carboxyl Terminus) familie van E3 ligases. Het wordt gekenmerkt door een sterk geconserveerd C-terminaal katalytisch domein bestaande uit 350 residuen. Veranderingen in de E6AP-functie, hetzij door activering of verlies, zijn gekoppeld aan verschillende menselijke ziekten. Verlies van E6AP-functie als gevolg van deletie, imprintingdefecten of mutaties in het UBE3A-gen gelegen in de 15q11–13 chromosoomregio wordt geassocieerd met Angelman-syndroom, een neurologische aandoening. De meeste natuurlijke mutaties in UBE3A leiden tot deleties die verkorte vormen van E6AP creëren waarbij het complete HECT-domein ontbreekt. Ongeveer 10% van de genetische veranderingen zijn echter puntmutaties binnen het E6AP-coderende gebied. Omgekeerd is duplicatie van het UBE3A-gen gesuggereerd als een bijdragende factor in sommige gevallen van autismespectrumstoornis. Het oncogene E6-eiwit van hoogrisico humane papillomavirussen (HPV16 en HPV18) kan UBE3A kapen, wat leidt tot onjuiste ubiquitinatie van specifieke cellulaire eiwitten, met name p53, wat significant is in de context van kankerontwikkeling. In neuronen resulteert verstoring van de UBE3A-functie in verhoogde Arc-expressie en een vermindering van AMPA-receptoren (AMPARs) bij excitatorische synapsen, wat potentieel bijdraagt aan de neurologische symptomen van Angelman-syndroom.

Gebruiksinformatie

Toepassing

WB

Verdunning

WB

1:1000

Reactiviteit

Human, Mouse, Rat, Monkey

Bron

Rabbit Monoclonal Antibody

MW

98 kDa

Opslagbuffer

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

Opslag
(Vanaf de datum van ontvangst)

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

Experimentele Methoden
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 965. 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.

Experimentele Methoden

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

965. 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.

Referenties

https://pubmed.ncbi.nlm.nih.gov/24273172/
https://pubmed.ncbi.nlm.nih.gov/8380895/

Toepassingsgegevens

WB

Gevalideerd door Selleck

Lane 1: K562
Lane 2: NIH/3T3
Lane 3: COS-7
Lane 4: A172