Datenblatt

Biologische Beschreibung

Spezifität	Phospho-Tau (Ser202/Thr205) Antibody [M2L8] erkennt endogene Mengen an Gesamt-Tau-Protein nur, wenn es sowohl an Ser202 als auch an Thr205 phosphoryliert ist.
Hintergrund	Phospho-Tau (Ser202/Thr205) bezieht sich auf Tau-Protein, das an Serin 202 und Threonin 205 phosphoryliert ist, zwei benachbarte Reste innerhalb der prolinreichen Region dieses Microtubule Associated Proteins. Tau ist ein intrinsisch ungeordnetes Protein, das die Assemblierung von Mikrotubuli stabilisiert und fördert. Die Phosphorylierung an Ser202/Thr205 induziert eine Konformationsänderung, die die N-terminale Region erweitert und den Abstand zwischen Mikrotubuli in Neuriten vergrößert. Diese Stellen werden von Kinasen wie Cdk5, GSK-3β, PKA und Casein-Kinase II phosphoryliert, wobei Cdk5 Thr205 bevorzugt vor Ser202 phosphoryliert und die Phosphorylierungsreihenfolge durch die Assoziation von Tau mit Mikrotubuli beeinflusst wird. Die Phosphorylierung an diesen Stellen reguliert die Mikrotubuli-Bindungsaffinität von Tau, wodurch im Allgemeinen seine Fähigkeit zur Stabilisierung von Mikrotubuli verringert und zur cytoskeletal Destabilisierung beigetragen wird. Eine Hyperphosphorylierung an Ser202/Thr205 ist ein Schlüsselereignis bei neurodegenerativen Erkrankungen, das zur Tau-Aggregation und zur Bildung neurofibrillärer Tangles führt, die charakteristisch für die Alzheimer-Krankheit und andere Tauopathien sind. Der Phosphorylierungszustand an diesen Resten wird auch durch verschiedene Phosphatasen moduliert, einschließlich PP2A, PP2B und PP5, die effektiver sind als PP1 bei der Dephosphorylierung von Tau an diesen Stellen. Diese Modifikationen verändern die Konformation von Tau, seine Mikrotubuli-Interaktionen und die zelluläre Lokalisation und spielen eine zentrale Rolle bei der Krankheitsentwicklung, indem sie die neuronale Funktion beeinträchtigen und die Tau-Pathologie fördern.

Spezifität

Phospho-Tau (Ser202/Thr205) Antibody [M2L8] erkennt endogene Mengen an Gesamt-Tau-Protein nur, wenn es sowohl an Ser202 als auch an Thr205 phosphoryliert ist.

Hintergrund

Phospho-Tau (Ser202/Thr205) bezieht sich auf Tau-Protein, das an Serin 202 und Threonin 205 phosphoryliert ist, zwei benachbarte Reste innerhalb der prolinreichen Region dieses Microtubule Associated Proteins. Tau ist ein intrinsisch ungeordnetes Protein, das die Assemblierung von Mikrotubuli stabilisiert und fördert. Die Phosphorylierung an Ser202/Thr205 induziert eine Konformationsänderung, die die N-terminale Region erweitert und den Abstand zwischen Mikrotubuli in Neuriten vergrößert. Diese Stellen werden von Kinasen wie Cdk5, GSK-3β, PKA und Casein-Kinase II phosphoryliert, wobei Cdk5 Thr205 bevorzugt vor Ser202 phosphoryliert und die Phosphorylierungsreihenfolge durch die Assoziation von Tau mit Mikrotubuli beeinflusst wird. Die Phosphorylierung an diesen Stellen reguliert die Mikrotubuli-Bindungsaffinität von Tau, wodurch im Allgemeinen seine Fähigkeit zur Stabilisierung von Mikrotubuli verringert und zur cytoskeletal Destabilisierung beigetragen wird. Eine Hyperphosphorylierung an Ser202/Thr205 ist ein Schlüsselereignis bei neurodegenerativen Erkrankungen, das zur Tau-Aggregation und zur Bildung neurofibrillärer Tangles führt, die charakteristisch für die Alzheimer-Krankheit und andere Tauopathien sind. Der Phosphorylierungszustand an diesen Resten wird auch durch verschiedene Phosphatasen moduliert, einschließlich PP2A, PP2B und PP5, die effektiver sind als PP1 bei der Dephosphorylierung von Tau an diesen Stellen. Diese Modifikationen verändern die Konformation von Tau, seine Mikrotubuli-Interaktionen und die zelluläre Lokalisation und spielen eine zentrale Rolle bei der Krankheitsentwicklung, indem sie die neuronale Funktion beeinträchtigen und die Tau-Pathologie fördern.

Nutzungsinformationen

Anwendung

WB

Verdünnung

WB

1:1000

Reaktivität

Human, Mouse, Rat

Quelle

Rabbit Monoclonal Antibody

MW

55-80 kDa

Lagerpuffer

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

Lagerung
(Ab dem Datum des Erhalts)

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

Experimentelle 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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( 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 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. (Exposure time of at least 120s is recommended)

Experimentelle 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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( 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

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. (Exposure time of at least 120s is recommended)

Referenzen

https://pubmed.ncbi.nlm.nih.gov/28784767/
https://pubmed.ncbi.nlm.nih.gov/29467609/

Anwendungsdaten

WB

Validiert von Selleck

Lane 1: Mouse brain, Lane 2: Mouse brain (λ phosphatase-treated), Lane 3: Mouse brain (KO Tau)