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Anti-Phospho-Synapsin-1 (Ser605) Rabbit Antibody [M6M7]

Catalog No.: F1303

Application: Reactivity: Human, Mouse, Rat

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
75-90 kDa

Positive Control	Mouse brain; Rat brain
Negative Control	Mouse lung

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 150s 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 150s is recommended)

Datasheet & SDS

Biological Description

Specificity
Phospho-Synapsin-1 (Ser605) Rabbit mAb detects endogenous levels of Synapsin-1 protein only when phosphorylated at Ser605 (corresponds to Ser603 in rat).

Subcellular Location
Cell projection, Cytoplasmic vesicle, Golgi apparatus, Synapse

Uniprot ID
P17600

Clone
M6M7

Synonym(s)
Synapsin-1, Brain protein 4.1, Synapsin I, SYN1

Background
Synapsin I (protein I) is a major neuron-specific phosphoprotein and a key endogenous substrate for both cAMP-dependent and Ca²⁺/calmodulin-dependent protein kinases. It is widely distributed in synapses throughout the central and peripheral nervous systems, where it is specifically associated with the cytoplasmic surface of synaptic vesicle membranes. The synapsin protein family consists of four homologous members: synapsins Ia and Ib (collectively referred to as synapsin I) and synapsins IIa and IIb (collectively referred to as synapsin II). Together, synapsins I and II account for approximately 9% of total synaptic vesicle protein. Synapsins I and II are primarily present in mature synapses, whereas synapsin III is expressed mainly during synapse development and at comparatively lower levels. Functionally, synapsin I plays a critical role in regulating neurotransmitter release. In neurons, it helps control the availability of synaptic vesicles for exocytosis. Phosphorylation at the Ser-9 residue (phospho-Ser9 synapsin I) causes the protein to dissociate from synaptic vesicles, a process essential for neurotransmitter release. Additionally, synapsin I contributes to synaptic plasticity by influencing both pre- and post-synaptic vesicle release. Genetically, mutations in the synapsin I gene have been linked to X-linked epilepsy with variable learning disabilities and behavioral disorders (XELBD), a neurological condition characterized by varying combinations of epilepsy, cognitive impairments, macrocephaly, and aggressive behavior. Ser605 is confirmed as a major phosphorylation site on Synapsin I, with in vivo evidence supporting its significance. Phosphorylation at Ser605 (alongside Ser568) by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) disrupts Synapsin I's ability to bundle actin filaments. This is likely a mechanism regulating the dynamic organization of the presynaptic cytoskeleton

Background

Synapsin I (protein I) is a major neuron-specific phosphoprotein and a key endogenous substrate for both cAMP-dependent and Ca²⁺/calmodulin-dependent protein kinases. It is widely distributed in synapses throughout the central and peripheral nervous systems, where it is specifically associated with the cytoplasmic surface of synaptic vesicle membranes. The synapsin protein family consists of four homologous members: synapsins Ia and Ib (collectively referred to as synapsin I) and synapsins IIa and IIb (collectively referred to as synapsin II). Together, synapsins I and II account for approximately 9% of total synaptic vesicle protein. Synapsins I and II are primarily present in mature synapses, whereas synapsin III is expressed mainly during synapse development and at comparatively lower levels. Functionally, synapsin I plays a critical role in regulating neurotransmitter release. In neurons, it helps control the availability of synaptic vesicles for exocytosis. Phosphorylation at the Ser-9 residue (phospho-Ser9 synapsin I) causes the protein to dissociate from synaptic vesicles, a process essential for neurotransmitter release. Additionally, synapsin I contributes to synaptic plasticity by influencing both pre- and post-synaptic vesicle release. Genetically, mutations in the synapsin I gene have been linked to X-linked epilepsy with variable learning disabilities and behavioral disorders (XELBD), a neurological condition characterized by varying combinations of epilepsy, cognitive impairments, macrocephaly, and aggressive behavior. Ser605 is confirmed as a major phosphorylation site on Synapsin I, with in vivo evidence supporting its significance. Phosphorylation at Ser605 (alongside Ser568) by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) disrupts Synapsin I's ability to bundle actin filaments. This is likely a mechanism regulating the dynamic organization of the presynaptic cytoskeleton

References
https://pubmed.ncbi.nlm.nih.gov/8430330/ https://pubmed.ncbi.nlm.nih.gov/8702879/

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
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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%