Gegevensblad

Biologische Beschrijving

Specificiteit	Caspase-1 Antibody [C2H20] detecteert endogene niveaus van totaal Caspase-1 eiwit.
Achtergrond	Caspase-1 (interleukine-1β converterend enzym, ICE), geclassificeerd als een inflammatoire cysteïne protease van de Caspase familie naast caspasen-4, -5, -11 en -12, wordt gesynthetiseerd als een inactief 45 kDa pro-enzym dat essentieel is voor aangeboren immuunresponsen. Het beschikt over een N-terminaal Caspase rekruteringsdomein (CARD) voor inflammasoomassemblage, een grote p20 subeenheid die het katalytische cysteïneresidu bevat, en een kleine p10 subeenheid, die samen een actief heterotetrameer (p20)₂(p10)₂ vormen door nabijheidsgeïnduceerde dimerisatie en autoproteolytische splitsing. Caspase-1 activeert binnen multiproteïne inflammasomen (NLRP3, AIM2, NLRC4/ASC) geactiveerd door pathogeen-geassocieerde moleculaire patronen of schadesignalen, waarbij specifiek pro-IL-1β en pro-IL-18 worden gesplitst in rijpe pro-inflammatoire cytokines die koorts, ontsteking en adaptieve immuniteit aansturen, terwijl gasdermin D wordt verwerkt om pyroptose uit te voeren via N-terminale porievorming, wat leidt tot lytische celdood en IL-1-afgifte. Caspase-1-deficiëntie vermindert selectief de cytokinematratie en verleent resistentie tegen endotoxische shock zonder Apoptosis te beïnvloeden.

Specificiteit

Caspase-1 Antibody [C2H20] detecteert endogene niveaus van totaal Caspase-1 eiwit.

Achtergrond

Caspase-1 (interleukine-1β converterend enzym, ICE), geclassificeerd als een inflammatoire cysteïne protease van de Caspase familie naast caspasen-4, -5, -11 en -12, wordt gesynthetiseerd als een inactief 45 kDa pro-enzym dat essentieel is voor aangeboren immuunresponsen. Het beschikt over een N-terminaal Caspase rekruteringsdomein (CARD) voor inflammasoomassemblage, een grote p20 subeenheid die het katalytische cysteïneresidu bevat, en een kleine p10 subeenheid, die samen een actief heterotetrameer (p20)₂(p10)₂ vormen door nabijheidsgeïnduceerde dimerisatie en autoproteolytische splitsing. Caspase-1 activeert binnen multiproteïne inflammasomen (NLRP3, AIM2, NLRC4/ASC) geactiveerd door pathogeen-geassocieerde moleculaire patronen of schadesignalen, waarbij specifiek pro-IL-1β en pro-IL-18 worden gesplitst in rijpe pro-inflammatoire cytokines die koorts, ontsteking en adaptieve immuniteit aansturen, terwijl gasdermin D wordt verwerkt om pyroptose uit te voeren via N-terminale porievorming, wat leidt tot lytische celdood en IL-1-afgifte. Caspase-1-deficiëntie vermindert selectief de cytokinematratie en verleent resistentie tegen endotoxische shock zonder Apoptosis te beïnvloeden.

Gebruiksinformatie

Toepassing

WB, IP

Verdunning

WB	IP
1:1000	1:200

Reactiviteit

Mouse

Bron

Rabbit Monoclonal Antibody

MW

48 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. 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. ( 0.45 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.

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.

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. ( 0.45 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.

Referenties

https://pubmed.ncbi.nlm.nih.gov/33116753/
https://pubmed.ncbi.nlm.nih.gov/19221555/

Toepassingsgegevens

WB

Gevalideerd door Selleck

Lane 1: A20, Lane 2: EL4, Lane 3: J774A.1