Datasheet

Biological Description

Specificity	LIM Kinase 1 Rabbit mAb detects total endogenous levels of LIM Kinase 1 protein.
Background	The organization of the actin cytoskeleton is essential for numerous cellular processes, including survival, differentiation, proliferation, cytokinesis, apoptosis, and motility. LIM kinase 1 (LIMK1) and its homolog, LIM kinase 2 (LIMK2), are serine protein kinases that regulate actin dynamics by phosphorylating their substrate proteins, such as actin depolymerizing factor (ADF) and cofilin, thereby controlling actin polymerization. The activity of LIMK proteins is modulated by members of the Rho GTPase family, including Rho, Rac, and Cdc42, through their downstream effectors, such as Rho-associated kinase (ROCK) and p21-activated kinases (PAK1 and PAK4). These kinases phosphorylate LIMK1 at threonine 508 (Thr508) within the activation loop of the kinase domain, leading to its activation. Similarly, both PAK1 and ROCK phosphorylate LIMK1 and LIMK2 at conserved residues, Thr508 and Thr505, respectively, enhancing their activity. LIM kinases, along with the phosphatase SSH-1, are key regulators of cofilin-mediated protrusion dynamics. LIMK1 promotes cell migration by facilitating lamellipodium formation during the initial stages of cellular response, while SSH-1 directs migration by limiting protrusion formation to a single direction, ensuring coordinated movement. LIMK1 and LIMK2 share a conserved domain structure comprising two N-terminal LIM domains, a PDZ domain, a serine/proline-rich region, and a C-terminal kinase domain. Beyond their role in actin regulation, LIM kinases are also integral to the coordination of actin and microtubule dynamics. LIM kinases interact with both actin and tubulin, with ROCK-mediated phosphorylation of LIMK1 promoting actin interaction while reducing its binding to tubulin. Overexpression of LIMK1 destabilizes microtubules, a process dependent on its kinase activity. Conversely, inhibition of ROCK stabilizes a trimeric complex involving LIMK1, TPPP1, and HDAC6 (histone deacetylase 6), influencing tubulin acetylation and microtubule stability. LIMK overexpression can result in tubulin acetylation and microtubule stabilization, whereas TPPP1 overexpression leads to decreased cofilin phosphorylation and disruption of stress fibers.

Specificity

LIM Kinase 1 Rabbit mAb detects total endogenous levels of LIM Kinase 1 protein.

Background

The organization of the actin cytoskeleton is essential for numerous cellular processes, including survival, differentiation, proliferation, cytokinesis, apoptosis, and motility. LIM kinase 1 (LIMK1) and its homolog, LIM kinase 2 (LIMK2), are serine protein kinases that regulate actin dynamics by phosphorylating their substrate proteins, such as actin depolymerizing factor (ADF) and cofilin, thereby controlling actin polymerization. The activity of LIMK proteins is modulated by members of the Rho GTPase family, including Rho, Rac, and Cdc42, through their downstream effectors, such as Rho-associated kinase (ROCK) and p21-activated kinases (PAK1 and PAK4). These kinases phosphorylate LIMK1 at threonine 508 (Thr508) within the activation loop of the kinase domain, leading to its activation. Similarly, both PAK1 and ROCK phosphorylate LIMK1 and LIMK2 at conserved residues, Thr508 and Thr505, respectively, enhancing their activity. LIM kinases, along with the phosphatase SSH-1, are key regulators of cofilin-mediated protrusion dynamics. LIMK1 promotes cell migration by facilitating lamellipodium formation during the initial stages of cellular response, while SSH-1 directs migration by limiting protrusion formation to a single direction, ensuring coordinated movement. LIMK1 and LIMK2 share a conserved domain structure comprising two N-terminal LIM domains, a PDZ domain, a serine/proline-rich region, and a C-terminal kinase domain. Beyond their role in actin regulation, LIM kinases are also integral to the coordination of actin and microtubule dynamics. LIM kinases interact with both actin and tubulin, with ROCK-mediated phosphorylation of LIMK1 promoting actin interaction while reducing its binding to tubulin. Overexpression of LIMK1 destabilizes microtubules, a process dependent on its kinase activity. Conversely, inhibition of ROCK stabilizes a trimeric complex involving LIMK1, TPPP1, and HDAC6 (histone deacetylase 6), influencing tubulin acetylation and microtubule stability. LIMK overexpression can result in tubulin acetylation and microtubule stabilization, whereas TPPP1 overexpression leads to decreased cofilin phosphorylation and disruption of stress fibers.

Usage Information

Application

WB

Dilution

WB

1:1000

Reactivity

Human

Source

Rabbit

MW

73 kDa

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

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)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 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.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 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 1247. 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 300s is recommended)

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)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

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.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

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

1247. 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 300s is recommended)

References

Application Data

WB

Validated by Selleck

Lane 1: HAP1
Lane 2: HAP1 (KO LIMK1)
Lane 3: SH SY5Y