Phospho-PERK (Thr980) Antibody (Rabbit mAb) [E9N5]

CatNo: F2713

Application: Reactivity: Rat

Lane 1: AR42J, Lane 2: AR42J (thapsigargin (Tg), 1 μM, 20 min)

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

キーポイント

WB
SDS-PAGE の分離ゲルの推奨濃度：5%。

使用情報

Dilution
WB1:1000

Application
WB

Source
Rabbit Monoclonal Antibody

Reactivity
Rat

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
170 kDa

ポジティブコントロール	AR42J cells (Thapsigargin, 20 min); RSC96 cells (Thapsigargin, 300nM 2 h); RSC96 cells (Tunicamycin, 4 µg/mL, 2 h)
ネガティブコントロール	RSC96 cells; AR42J cells

プロトコール

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 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 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 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

生物学的記述

Specificity
Phospho-PERK (Thr980) Antibody (Rabbit mAb) [E9N5] detects endogenous levels of total PERK protein only when it is phosphorylated at Thr980.

タンパク質の局在
小胞体、細胞内膜系

Uniprot ID
Q9NZJ5

Clone
E9N5

Synonym(s)
Eukaryotic translation initiation factor 2-alpha kinase 3; Pancreatic eIF2-alpha kinase; PRKR-like endoplasmic reticulum kinase; Protein tyrosine kinase EIF2AK3, Eif2ak3; PEK; Perk

Background
Phospho-PERK (Thr980) designates the activated form of protein kinase–like endoplasmic reticulum kinase (PERK/EIF2AK3) in which the conserved threonine residue within the activation loop of the cytoplasmic kinase domain is autophosphorylated, marking engagement of the PERK arm of the unfolded protein response and coupling ER stress to translational control and downstream signaling. PERK is a type I ER transmembrane serine/threonine kinase with a luminal stress-sensing domain that binds the chaperone BiP under basal conditions and a cytosolic kinase region that dimerizes and oligomerizes upon BiP release and accumulation of misfolded proteins, creating a platform for activation-loop phosphorylation at Thr980 and related residues. Autophosphorylation at Thr980 stabilizes the active conformation of the kinase domain and enables efficient phosphorylation of eIF2α on Ser51, which reduces global cap-dependent translation while permitting selective translation of stress-responsive mRNAs, including ATF4, thereby decreasing ER protein load and initiating transcriptional programs for amino acid metabolism, antioxidant defense, and apoptosis regulators. Thr980 phosphorylation is therefore widely used as a biochemical surrogate for PERK activity, and phospho-specific antibodies raised against a phosphopeptide encompassing this residue detect increased P‑Thr980 upon treatment with ER stressors such as tunicamycin or thapsigargin in multiple cell types. PERK signaling contributes to G1 growth arrest through loss of cyclin D1 and promotes adaptation or cell death depending on stress intensity and duration, and defects in PERK (including loss-of-function mutations in EIF2AK3) underlie Wolcott–Rallison syndrome, emphasizing the importance of regulated PERK activation in β cell survival and systemic homeostasis.

Background

Phospho-PERK (Thr980) designates the activated form of protein kinase–like endoplasmic reticulum kinase (PERK/EIF2AK3) in which the conserved threonine residue within the activation loop of the cytoplasmic kinase domain is autophosphorylated, marking engagement of the PERK arm of the unfolded protein response and coupling ER stress to translational control and downstream signaling. PERK is a type I ER transmembrane serine/threonine kinase with a luminal stress-sensing domain that binds the chaperone BiP under basal conditions and a cytosolic kinase region that dimerizes and oligomerizes upon BiP release and accumulation of misfolded proteins, creating a platform for activation-loop phosphorylation at Thr980 and related residues. Autophosphorylation at Thr980 stabilizes the active conformation of the kinase domain and enables efficient phosphorylation of eIF2α on Ser51, which reduces global cap-dependent translation while permitting selective translation of stress-responsive mRNAs, including ATF4, thereby decreasing ER protein load and initiating transcriptional programs for amino acid metabolism, antioxidant defense, and apoptosis regulators. Thr980 phosphorylation is therefore widely used as a biochemical surrogate for PERK activity, and phospho-specific antibodies raised against a phosphopeptide encompassing this residue detect increased P‑Thr980 upon treatment with ER stressors such as tunicamycin or thapsigargin in multiple cell types. PERK signaling contributes to G1 growth arrest through loss of cyclin D1 and promotes adaptation or cell death depending on stress intensity and duration, and defects in PERK (including loss-of-function mutations in EIF2AK3) underlie Wolcott–Rallison syndrome, emphasizing the importance of regulated PERK activation in β cell survival and systemic homeostasis.

References
[1]Cui W, et al. Acta Crystallogr D Biol Crystallogr. 2011 May;67(Pt 5):423-8. [2] Qi L, et al. Methods Enzymol. 2011;490:137-46.

PVDFメンブレン孔径参考表

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%