Scheda tecnica

Descrizione biologica

Specificità	A1/Bfl-1 Antibody [D21F14] riconosce i livelli endogeni della proteina A1/Bfl-1 totale.
Contesto	La proteina A1 correlata a Bcl-2 (nota anche come Bfl-1 o BCL2A1) è un membro anti-apoptotico della famiglia Bcl-2. La sua espressione si trova prevalentemente nelle cellule ematopoietiche, sebbene sia stata rilevata anche in tessuti non ematopoietici come il polmone e nelle cellule endoteliali. A1/Bfl-1 è rapidamente sovraregolata dall'attivazione di NF-κB in risposta a vari stimoli, inclusi TNF-α, IL-1β, legame CD40, esteri di forbolo e LPS. A1/Bfl-1 inibisce l'apoptosi legandosi e neutralizzando i membri pro-apoptotici della famiglia Bcl-2, come Bid e Bim, prevenendo così il rilascio di citocromo c dai mitocondri. È interessante notare che, in condizioni specifiche che coinvolgono la stimolazione del TNF e il trattamento con cicloeximide nelle cellule pro-B FL5.12, l'A1/Bfl-1 umano può essere clivato proteoliticamente dalla μ-calpaina o da una proteasi simile, convertendolo in un fattore pro-apoptotico. A1/Bfl-1 è stato implicato nell'autoimmunità, così come nella progressione e nella resistenza alla terapia dei tumori ematologici e solidi dove la sua espressione può essere disregolata. Oltre al suo ruolo nell'apoptosi, A1/Bfl-1, come altre proteine anti-apoptotiche come Bcl-2, Mcl-1 e Bcl-xL, può legarsi a Beclin1. Questa interazione inibisce l'autofagia, potenzialmente interrompendo la formazione dei complessi Beclin1-hVps34.

Specificità

A1/Bfl-1 Antibody [D21F14] riconosce i livelli endogeni della proteina A1/Bfl-1 totale.

Contesto

La proteina A1 correlata a Bcl-2 (nota anche come Bfl-1 o BCL2A1) è un membro anti-apoptotico della famiglia Bcl-2. La sua espressione si trova prevalentemente nelle cellule ematopoietiche, sebbene sia stata rilevata anche in tessuti non ematopoietici come il polmone e nelle cellule endoteliali. A1/Bfl-1 è rapidamente sovraregolata dall'attivazione di NF-κB in risposta a vari stimoli, inclusi TNF-α, IL-1β, legame CD40, esteri di forbolo e LPS. A1/Bfl-1 inibisce l'apoptosi legandosi e neutralizzando i membri pro-apoptotici della famiglia Bcl-2, come Bid e Bim, prevenendo così il rilascio di citocromo c dai mitocondri. È interessante notare che, in condizioni specifiche che coinvolgono la stimolazione del TNF e il trattamento con cicloeximide nelle cellule pro-B FL5.12, l'A1/Bfl-1 umano può essere clivato proteoliticamente dalla μ-calpaina o da una proteasi simile, convertendolo in un fattore pro-apoptotico. A1/Bfl-1 è stato implicato nell'autoimmunità, così come nella progressione e nella resistenza alla terapia dei tumori ematologici e solidi dove la sua espressione può essere disregolata. Oltre al suo ruolo nell'apoptosi, A1/Bfl-1, come altre proteine anti-apoptotiche come Bcl-2, Mcl-1 e Bcl-xL, può legarsi a Beclin1. Questa interazione inibisce l'autofagia, potenzialmente interrompendo la formazione dei complessi Beclin1-hVps34.

Informazioni sullutilizzo

Applicazione

WB, IP

Diluizione

WB	IP
1:1000	1:200

Reattività

Human

Fonte

Rabbit Monoclonal Antibody

MW

18 kDa

Tampone di conservazione

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

Conservazione
(Dalla data di ricevimento)

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

Metodi sperimentali
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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. ( 0.22 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 1173. 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.

Metodi sperimentali

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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. ( 0.22 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

1173. 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.

Riferimenti

https://pubmed.ncbi.nlm.nih.gov/22342458/

Dati di applicazione

WB

Validato da Selleck

Lane 1: REC-1
Lane 2: SU-DHL-1
Lane 3: SK-MEL-30