STIM2 C-terminal Antibody (Rabbit mAb) [N9G10] Datasheet

Biological Description

Specificity	STIM2 C-terminal Antibody (Rabbit mAb) [N9G10] detects endogenous levels of total STIM2 C-terminal protein.
Background	STIM2, a paralog of STIM1 within the stromal interaction molecule family of endoplasmic reticulum calcium sensors, shares substantial sequence identity with STIM1 and retains the same overall domain architecture, including a luminal EF-hand and sterile alpha motif that sense ER calcium, a single transmembrane segment, and a long cytosolic C-terminal strand that couples to plasma membrane Orai1 channels. The C-terminal region contains the STIM-Orai activating region, a coiled-coil domain that binds directly to Orai1 and triggers channel opening upon store depletion, and this SOAR domain in STIM2 binds Orai1 with lower affinity than the equivalent STIM1 region, making STIM2 a comparatively weaker and slower activator of store-operated calcium entry. Further along the C-terminus, STIM2 carries a lysine-rich polybasic domain that binds phosphatidylinositol 4,5-bisphosphate in the plasma membrane, and this domain shows higher affinity for PI(4,5)P2-containing membranes than the corresponding STIM1 region, a property that positions STIM2-containing ER tubules closer to the plasma membrane and sensitizes the protein to smaller decreases in ER calcium concentration. Calmodulin binding to this same C-terminal polybasic domain is calcium- and PI(4,5)P2-dependent, and calmodulin engagement inhibits the domain's interaction with plasma membrane lipids, meaning that rising cytosolic calcium feeds back through calmodulin to downregulate STIM2-mediated ER-plasma membrane contact formation. The combination of a lower-affinity EF-hand calcium sensor, a weaker Orai1-coupling SOAR domain, and a higher-affinity lipid-binding polybasic region gives STIM2 a distinct operational profile from STIM1: rather than driving the large, rapid calcium influx associated with immune cell activation, STIM2 operates closer to resting ER calcium levels and functions primarily to stabilize basal cytosolic and ER calcium concentrations. This basal-regulatory role extends into synaptic contexts, where STIM2-mediated control of resting calcium levels supports synaptic structure and stability, and disruption of this regulatory function is implicated in the calcium dyshomeostasis observed in Alzheimer's disease. STIM2 additionally functions through a store-independent mode involving interaction with and dissociation from calmodulin, giving the protein two distinct modes of coupling to downstream calcium channels beyond classical store depletion. The luminal domain of STIM2 also carries cysteine residues subject to nitrosative modification, and thiol-based stabilization of this region suppresses both basal and store-operated calcium entry, indicating that redox regulation acts as an additional control layer on STIM2 activity distinct from its C-terminal Orai1- and lipid-coupling mechanisms.

Specificity

STIM2 C-terminal Antibody (Rabbit mAb) [N9G10] detects endogenous levels of total STIM2 C-terminal protein.

Background

STIM2, a paralog of STIM1 within the stromal interaction molecule family of endoplasmic reticulum calcium sensors, shares substantial sequence identity with STIM1 and retains the same overall domain architecture, including a luminal EF-hand and sterile alpha motif that sense ER calcium, a single transmembrane segment, and a long cytosolic C-terminal strand that couples to plasma membrane Orai1 channels. The C-terminal region contains the STIM-Orai activating region, a coiled-coil domain that binds directly to Orai1 and triggers channel opening upon store depletion, and this SOAR domain in STIM2 binds Orai1 with lower affinity than the equivalent STIM1 region, making STIM2 a comparatively weaker and slower activator of store-operated calcium entry. Further along the C-terminus, STIM2 carries a lysine-rich polybasic domain that binds phosphatidylinositol 4,5-bisphosphate in the plasma membrane, and this domain shows higher affinity for PI(4,5)P2-containing membranes than the corresponding STIM1 region, a property that positions STIM2-containing ER tubules closer to the plasma membrane and sensitizes the protein to smaller decreases in ER calcium concentration. Calmodulin binding to this same C-terminal polybasic domain is calcium- and PI(4,5)P2-dependent, and calmodulin engagement inhibits the domain's interaction with plasma membrane lipids, meaning that rising cytosolic calcium feeds back through calmodulin to downregulate STIM2-mediated ER-plasma membrane contact formation. The combination of a lower-affinity EF-hand calcium sensor, a weaker Orai1-coupling SOAR domain, and a higher-affinity lipid-binding polybasic region gives STIM2 a distinct operational profile from STIM1: rather than driving the large, rapid calcium influx associated with immune cell activation, STIM2 operates closer to resting ER calcium levels and functions primarily to stabilize basal cytosolic and ER calcium concentrations. This basal-regulatory role extends into synaptic contexts, where STIM2-mediated control of resting calcium levels supports synaptic structure and stability, and disruption of this regulatory function is implicated in the calcium dyshomeostasis observed in Alzheimer's disease. STIM2 additionally functions through a store-independent mode involving interaction with and dissociation from calmodulin, giving the protein two distinct modes of coupling to downstream calcium channels beyond classical store depletion. The luminal domain of STIM2 also carries cysteine residues subject to nitrosative modification, and thiol-based stabilization of this region suppresses both basal and store-operated calcium entry, indicating that redox regulation acts as an additional control layer on STIM2 activity distinct from its C-terminal Orai1- and lipid-coupling mechanisms.

Usage Information

Application

WB

Dilution

WB

1:1000

Reactivity

Human

Source

Rabbit Monoclonal Antibody

MW

84 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

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)，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) , 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) , 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), 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 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 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) , 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) , 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 primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), 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.

References

https://pubmed.ncbi.nlm.nih.gov/24044355/
https://pubmed.ncbi.nlm.nih.gov/32576932/

Application Data

WB

Validated by Selleck

Lane 1: 293T, Lane 2: Hela, Lane 3: Jurkat