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Anti-Phospho-Tau (Ser202/Thr205) Rabbit Antibody [M2L8]

Catalog No.: F3533

Application: Reactivity: Human, Mouse, Rat

Experiment Essentials

WB
Exposure time of at least 120s is recommended.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat

Source
Rabbit

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

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
55-80 kDa

Positive Control	Mouse brain; hTau Tg2508 Mouse brain tissue
Negative Control	Mouse liver

Exprimental Methods

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)

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)

Datasheet & SDS

Biological Description

Specificity
Phospho-Tau (Ser202/Thr205) Rabbit mAb recognizes endogenous levels of total Tau protein only when phosphorylated at both Ser202 and Thr205.

Subcellular Location
Cell membrane, Cell projection, Cytoplasm, Cytoskeleton, Membrane, Microtubule, Secreted

Uniprot ID
P10636

Clone
M2L8

Synonym(s)
Microtubule-associated protein tau; Neurofibrillary tangle protein; Paired helical filament-tau (PHF-tau); MAPT; MAPTL; MTBT1; TAU

Background
Phospho-Tau (Ser202/Thr205) refers to tau protein phosphorylated at serine 202 and threonine 205, two adjacent residues within the proline-rich region of this microtubule-associated protein. Tau is an intrinsically disordered protein that stabilizes and promotes the assembly of microtubules. Phosphorylation at Ser202/Thr205 induces a conformational change that extends the N-terminal region and increases the spacing between microtubules in neurites. These sites are phosphorylated by kinases such as Cdk5, GSK-3β, PKA, and casein kinase II, with Cdk5 preferentially phosphorylating Thr205 before Ser202, and the phosphorylation order influenced by tau’s association with microtubules. Phosphorylation at these sites regulates tau’s microtubule-binding affinity, generally reducing its ability to stabilize microtubules and contributing to cytoskeletal destabilization. Hyperphosphorylation at Ser202/Thr205 is a key event in neurodegenerative diseases, leading to tau aggregation and the formation of neurofibrillary tangles, which are characteristic of Alzheimer’s disease and other tauopathies. The phosphorylation state at these residues is also modulated by various phosphatases, including PP2A, PP2B, and PP5, which are more effective than PP1 at dephosphorylating tau at these sites. These modifications alter tau’s conformation, microtubule interactions, and cellular localization, and play a central role in disease progression by impairing neuronal function and promoting tau pathology.

Background

Phospho-Tau (Ser202/Thr205) refers to tau protein phosphorylated at serine 202 and threonine 205, two adjacent residues within the proline-rich region of this microtubule-associated protein. Tau is an intrinsically disordered protein that stabilizes and promotes the assembly of microtubules. Phosphorylation at Ser202/Thr205 induces a conformational change that extends the N-terminal region and increases the spacing between microtubules in neurites. These sites are phosphorylated by kinases such as Cdk5, GSK-3β, PKA, and casein kinase II, with Cdk5 preferentially phosphorylating Thr205 before Ser202, and the phosphorylation order influenced by tau’s association with microtubules. Phosphorylation at these sites regulates tau’s microtubule-binding affinity, generally reducing its ability to stabilize microtubules and contributing to cytoskeletal destabilization. Hyperphosphorylation at Ser202/Thr205 is a key event in neurodegenerative diseases, leading to tau aggregation and the formation of neurofibrillary tangles, which are characteristic of Alzheimer’s disease and other tauopathies. The phosphorylation state at these residues is also modulated by various phosphatases, including PP2A, PP2B, and PP5, which are more effective than PP1 at dephosphorylating tau at these sites. These modifications alter tau’s conformation, microtubule interactions, and cellular localization, and play a central role in disease progression by impairing neuronal function and promoting tau pathology.

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

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%