ホームページ 一次抗体 Phospho-4E-BP1 (Thr70) Rabbit mAb For research use only.
Not for use in humans.

Phospho-4E-BP1 (Thr70) Rabbit mAb

Catalog No.: F0851

  • Lane 1: HeLa (serum starved)
    Lane 2: HeLa (serum starved; Insulin, 1 μM, 30 min)
    Lane 3: HeLa (serum starved; Insulin and λ phosphatase-treated)
サイズ （液体） 価格(税別) 在庫状況
JPY 23500 国内在庫なし(納期7~10日)
JPY 35500 国内在庫なし(納期7~10日)
JPY 53500 お問い合わせ

代表番号: 045-509-1970|電子メール：sales@selleck.co.jp
よく尋ねられる質問

カスタマーフィードバック(1)

WB

キーポイント

タンパク質の局在：細胞質，細胞核。
WB
RIPA/NP-40 Lysis Buffer バッファーでのライセート調製を推奨します。
SDS-PAGE の分離ゲルの推奨濃度：20%。
転写条件（ウェット）: 200 mA, 60 min，Recommended to use 0.22 μm PVDF 膜の使用をお勧めします。

使用情報

Dilution
1:1000
1:50
Application
WB, IP
Source
Rabbit
Reactivity
Human, Monkey
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
15-20 15-20 kDa
*なぜ予測分子量と実際の分子量が異なるのか？
下記の原因により、実際の分子量が予測と異なる：タンパク質の翻訳後修飾（リン酸化/糖鎖付加），スプライシングバリアント，イソフォーム，相対的な電荷，ポリマー。
ポジティブコントロール Hela (serum starved); Hela (serum starved; insulin-treated, 1 μM, 30 min)
ネガティブコントロール HeLa (λ phosphatase-treated)

サンプル処理データの例

サンプル 処理状況
Hela Serum starvation
Hela Serum starvation + Insulin treatment (1 μM, 30 min)
クリックして、さらに多くのサンプルデータを表示

*異なるヒト由来細胞や組織における発現量の予測については、以下をご参照ください: http://www.proteinatlas.org

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.
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: 20%. 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. ( 0.22 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.
 
IF
Experimental Protocol：
 
Specimen Preparation 
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.
NOTE: Paraformaldehyde is toxic, use only in a fume hood.
2. Fix cells for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining.
 
Immunostaining
1. Add theblocking buffer and incubate for 60 min at RT.
2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Mount slides usingmounting medium with DAPI and cover with coverslips.
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.
 
IHC
Experimental Protocol：
 
Deparaffinization/Rehydration
1. Deparaffinize/hydrate sections:
2. Incubate sections in three washes of xylene for 5 min each.
3. Incubate sections in two washes of 100% ethanol for 10 min each.
4. Incubate sections in two washes of 95% ethanol for 10 min each.
5. Wash sections two times in dH2O for 5 min each.
6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
 
Staining
1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.
 

Datasheet & SDS

生物学的記述

Specificity

Phospho-4E-BP1 (Thr70) Rabbit mAb recognizes endogenous levels of 4E-BP1 protein only when phosphorylated at Ser70.
Uniprot ID
Q13541
Clone
K18C18
Background

Translation repressor protein 4E-BP1 (also known as PHAS-1) functions by inhibiting cap-dependent translation through its binding to the translation initiation factor eIF4E. The interaction is disrupted upon hyperphosphorylation of 4E-BP1, leading to the activation of cap-dependent translation. The activity of 4E-BP1 is regulated by both the PI3 kinase/Akt pathway and FRAP/mTOR kinase. In vivo, multiple residues of 4E-BP1 undergo phosphorylation. While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not impede the binding of 4E-BP1 to eIF4E, it is believed to prepare 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70.
References

技術サポート

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