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Anti-Phospho-Hsp27 (Ser82) Rabbit Antibody [M1P19]

Catalog No.: F3764

Application: Reactivity: Mouse, Rat, Human

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 - 1:2000 IP1:75

Application
WB, IP

Reactivity
Mouse, Rat, Human

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 Observed MW
23 kDa 27 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight.

Positive Control	Mouse heart; Rat heart; HeLa (with 44°C heat shock treatment)
Negative Control

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, 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 ( 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.

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, 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 ( 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.

Datasheet & SDS

Biological Description

Specificity
Anti-Phospho-Hsp27 (Ser82) Rabbit Antibody [M1P19] detects endogenous levels of total Phospho-HSP27 (Ser82) protein only when phosphorylated at Ser82.

Subcellular Location
Cytoplasm, Cytoskeleton, Nucleus

Uniprot ID
P04792

Clone
M1P19

Synonym(s)
HSP27, HSP28, HSPB1, Heat shock protein beta-1, HspB1, 28 kDa heat shock protein, Estrogen-regulated 24 kDa protein, Heat shock 27 kDa protein, Heat shock protein family B member 1, Stress-responsive protein 27, HSP 27, SRP27

Background
Heat shock protein 27 (HSP27) is a member of the small heat shock protein (sHSP) family (12–43 kDa) and exhibits a wide range of cellular functions. It acts as a molecular chaperone, an antioxidant, a regulator of apoptosis, and a mediator of actin cytoskeletal remodeling. Like other small HSPs, HSP27 contains a conserved α-crystallin domain located within its C-terminal region. Under oxidative stress, HSP27 protects cells by lowering intracellular levels of reactive oxygen species (ROS), achieved through enhancing glutathione production and reducing free iron availability. It also exhibits strong anti-apoptotic activity, influencing both mitochondria-dependent and independent apoptotic pathways. For example, HSP27 binds to DAXX during Fas–FasL–mediated apoptosis, preventing DAXX from associating with ASK1. Additionally, it interacts with Bax and cytochrome c, thereby suppressing mitochondrial apoptosis and blocking caspase-dependent cell death. HSP27 is expressed at basal levels in most cells and tissues, where it typically exists as large oligomeric complexes. Upon exposure to stress, its expression rises significantly, enhancing cellular resistance to damaging conditions. The protein is subject to stress-induced phosphorylation at Ser15, Ser78, and Ser82 in humans (Ser15 and Ser86 in rodents), mediated by MAPKAP kinase 2/3 downstream of the p38 MAPK signaling pathway. Phosphorylation not only influences its oligomeric state but also regulates its interactions with partner proteins. In neutrophils, HSP27 forms a complex with AKT and MAPKAP kinase 2, which suppresses constitutive apoptosis and promotes an inflammatory response. Stress-induced phosphorylation of HSP27 disrupts this complex, leading to dissociation from AKT and restoration of neutrophil apoptosis. Notably, the effects of phosphorylated HSP27 are highly context-dependent, varying with cell type and signaling environment.

Background

Heat shock protein 27 (HSP27) is a member of the small heat shock protein (sHSP) family (12–43 kDa) and exhibits a wide range of cellular functions. It acts as a molecular chaperone, an antioxidant, a regulator of apoptosis, and a mediator of actin cytoskeletal remodeling. Like other small HSPs, HSP27 contains a conserved α-crystallin domain located within its C-terminal region. Under oxidative stress, HSP27 protects cells by lowering intracellular levels of reactive oxygen species (ROS), achieved through enhancing glutathione production and reducing free iron availability. It also exhibits strong anti-apoptotic activity, influencing both mitochondria-dependent and independent apoptotic pathways. For example, HSP27 binds to DAXX during Fas–FasL–mediated apoptosis, preventing DAXX from associating with ASK1. Additionally, it interacts with Bax and cytochrome c, thereby suppressing mitochondrial apoptosis and blocking caspase-dependent cell death. HSP27 is expressed at basal levels in most cells and tissues, where it typically exists as large oligomeric complexes. Upon exposure to stress, its expression rises significantly, enhancing cellular resistance to damaging conditions. The protein is subject to stress-induced phosphorylation at Ser15, Ser78, and Ser82 in humans (Ser15 and Ser86 in rodents), mediated by MAPKAP kinase 2/3 downstream of the p38 MAPK signaling pathway. Phosphorylation not only influences its oligomeric state but also regulates its interactions with partner proteins. In neutrophils, HSP27 forms a complex with AKT and MAPKAP kinase 2, which suppresses constitutive apoptosis and promotes an inflammatory response. Stress-induced phosphorylation of HSP27 disrupts this complex, leading to dissociation from AKT and restoration of neutrophil apoptosis. Notably, the effects of phosphorylated HSP27 are highly context-dependent, varying with cell type and signaling environment.

References
https://pubmed.ncbi.nlm.nih.gov/22564335/

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

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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%