Phospho-GEF-H1 (Ser886) Antibody [E24J19]

Application: Reactivity: Human, Mouse, Rat

Lane 1: HT-29, Lane 2: HT-29 (thymidine, 2 mM, 16 h)

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat

Source
Rabbit Monoclonal Antibody

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
120 kDa

Positive Control	HT-29 cells (Thymidine, 2 mM, 16 h; Nocodazole, 10 nM, 24 h); 293T cells
Negative Control	HT-29 cells

Experimental 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes. 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) , 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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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: 5%. 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 primary antibody dilution buffer 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.

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 or lyse it by sonication on ice, then incubate on ice for 30 minutes.

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) , 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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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: 5%. 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 primary antibody dilution buffer 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
Phospho-GEF-H1 (Ser886) Antibody (Rabbit mAb) [E24J19] detects endogenous levels of total GEF-H1 protein only when it is phosphorylated at Ser886.

Subcellular Location
Cell junction, Cell membrane, Cell projection, Cytoplasm, Cytoskeleton, Golgi apparatus, Tight junction

Uniprot ID
Q92974

Clone
E24J19

Synonym(s)
Rho guanine nucleotide exchange factor 2, Guanine nucleotide exchange factor H1 (GEF‑H1), Microtubule‑regulated Rho‑GEF, Proliferating cell nucleolar antigen p40, ARHGEF2, KIAA0651, LFP40

Background
Phospho-GEF-H1 at Ser886 denotes the activated conformation of guanine nucleotide exchange factor H1 (GEF-H1/ARHGEF2), a DH-PH domain RhoA-specific GEF that cycles between microtubule-associated inhibition and cytoplasmic activation to drive actin dynamics and contractility. GEF-H1 possesses an N-terminal microtubule-binding C1 domain, central DH-PH GEF module catalyzing GDP/GTP exchange on RhoA, and C-terminal coiled-coil and 14-3-3 binding motifs including the RSLS motif harboring Ser886. Phosphorylation at Ser886 by Par1b/MARK2 kinase disrupts microtubule binding, releasing GEF-H1 into the cytoplasm where it engages RhoA to promote stress fiber formation, focal adhesion maturation, and actomyosin contractility; conversely, PP2A-mediated dephosphorylation restores microtubule association for sequestration. During cytokinesis, mitotic kinases like Aurora B phosphorylate Ser886 to localize GEF-H1 at the cleavage furrow, activating cortical RhoA for equatorial constriction and membrane ingression independent of centralspindlin-Ect2. Tight junction cingulin binds and inhibits GEF-H1 RhoGEF activity to maintain epithelial barrier integrity, while FcεRI signaling in mast cells activates it for granule exocytosis. Par1b targets three conserved serines near the C1 domain (including Ser886), converting dynamic microtubule turnover to stabilization in protrusions for cell polarization. Phospho-Ser886 GEF-H1 coordinates microtubule depolymerization with RhoA-mediated cytoskeletal remodeling in migration, division, and barrier function. Dysregulation promotes invasion in carcinomas and disrupts polarity in neurological disorders.

Background

Phospho-GEF-H1 at Ser886 denotes the activated conformation of guanine nucleotide exchange factor H1 (GEF-H1/ARHGEF2), a DH-PH domain RhoA-specific GEF that cycles between microtubule-associated inhibition and cytoplasmic activation to drive actin dynamics and contractility. GEF-H1 possesses an N-terminal microtubule-binding C1 domain, central DH-PH GEF module catalyzing GDP/GTP exchange on RhoA, and C-terminal coiled-coil and 14-3-3 binding motifs including the RSLS motif harboring Ser886. Phosphorylation at Ser886 by Par1b/MARK2 kinase disrupts microtubule binding, releasing GEF-H1 into the cytoplasm where it engages RhoA to promote stress fiber formation, focal adhesion maturation, and actomyosin contractility; conversely, PP2A-mediated dephosphorylation restores microtubule association for sequestration. During cytokinesis, mitotic kinases like Aurora B phosphorylate Ser886 to localize GEF-H1 at the cleavage furrow, activating cortical RhoA for equatorial constriction and membrane ingression independent of centralspindlin-Ect2. Tight junction cingulin binds and inhibits GEF-H1 RhoGEF activity to maintain epithelial barrier integrity, while FcεRI signaling in mast cells activates it for granule exocytosis. Par1b targets three conserved serines near the C1 domain (including Ser886), converting dynamic microtubule turnover to stabilization in protrusions for cell polarization. Phospho-Ser886 GEF-H1 coordinates microtubule depolymerization with RhoA-mediated cytoskeletal remodeling in migration, division, and barrier function. Dysregulation promotes invasion in carcinomas and disrupts polarity in neurological disorders.

References
https://pubmed.ncbi.nlm.nih.gov/21513698/ https://pubmed.ncbi.nlm.nih.gov/17488622/

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

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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%

Phospho-GEF-H1 (Ser886) Antibody (Rabbit mAb) [E24J19]