Phospho-Histone H2B (Ser14) Antibody [B13A9]

N.º de catálogo F1482

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Descripción biológica

Especificidad

Phospho-Histone H2B (Ser14) Antibody [B13A9] reconoce los niveles endógenos de la proteína histona H2B solo cuando está fosforilada en Ser14. El anticuerpo puede reaccionar de forma cruzada con una banda inespecífica de 30 kDa.
Antecedentes

La histona H2B es esencial para la dinámica de la cromatina y la reparación del ADN, y sus modificaciones postraduccionales desempeñan papeles cruciales en estos procesos. Entre estas, la fosforilación de la Serina 14 (Ser14) en la histona H2B es un marcador significativo en la respuesta celular a las roturas de doble cadena del ADN (DSB). La fosforilación en Ser14 (H2B-Ser14P) se asocia con las DSB y contribuye a la formación de focos inducidos por irradiación (IRIF), que son cruciales para la localización de los daños en el ADN y los factores de reparación en el sitio de la rotura. Si bien la γ-H2AX es vital para el reconocimiento inicial de las DSB y la remodelación de la cromatina, la formación de IRIF de H2B-Ser14P depende de la γ-H2AX y de las quinasas relacionadas con la fosfatidilinositol-3-OH quinasa (PIKK). Esta modificación es crucial para establecer un entorno de cromatina que apoye procesos eficaces de reparación del ADN.

Información de uso

Aplicación WB, IP Dilución
WB IP
1:1000 1:100
Reactividad Human, Mouse, Rat, Monkey, Bovine, Pig, Horse
Fuente Rabbit Monoclonal Antibody MW 14 Kda
Tampón de almacenamiento PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃
Almacenamiento
(Desde la fecha de recepción)		 –20°C (avoid freeze-thaw cycles), 2 years
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, Phosphatase 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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: 20%. 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 ( recommending 5% BSA 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
809. 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
Western Blotting
 
Sample preparation
 
1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane (For wet transfer: 150mA 50min).
 
Membrane Blocking and Antibody Incubations
 
a. Blocking
 
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.
 
b. Antibodies Incubation
 
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.
 
Detection of Proteins
 
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose in the imaging system.

Referencias

  • https://pubmed.ncbi.nlm.nih.gov/15197225/

Datos de aplicación

WB

Validado por Selleck

  • F1482-wb
    Lane 1: HL-60
    Lane 2: HL-60(etoposide-treated, 50 μM, 6 hr )
    Lane 3: HL-60(etoposide-treated, 50 μM, 6 hr; λ phosphatase treated)