ホームページ 一次抗体 Phospho-TRAF2 (Ser11) Rabbit mAb For research use only.
Not for use in humans.

Phospho-TRAF2 (Ser11) Rabbit mAb

Catalog No.: F1348

  • Lane 1: THP-1(TPA, 80 nM, overnight)
    Lane 2: THP-1(TPA, 80 nM, overnight; transfect poly(dA:dT), 0.004 U/ml, 7 hrs)
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

キーポイント

タンパク質の局在：細胞質。
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。

使用情報

Dilution
1:1000
Application
WB
Source
Rabbit
Reactivity
Human, Mouse
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
53 kDa
ポジティブコントロール THP-1 ( poly (dA:dT), 0.004 U/ml, 7h )
ネガティブコントロール

サンプル処理データの例

サンプル 処理状況
THP-1 TPA (80 nM, overnight)
THP-1 TPA (80 nM, overnight)+ poly(dA:dT) (0.004 U/mL; 7r ),
THP-1 TPA (80 nM, overnight)+LPS (1μg/mL, 0.25/1 h)
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
Western Blotting
 
Sample preparation
 
1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane (For wet transfer: 150mA 120min).
 
Membrane Blocking and Antibody Incubations
 
a. Blocking
 
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.
 
b. Antibodies Incubation
 
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.
 
Detection of Proteins
 
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic 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.
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: 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 ( 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-TRAF2 (Ser11) Rabbit mAb recognizes endogenous levels of TRAF2 protein only when phosphorylated at Ser11. "
Uniprot ID
Q12933
Clone
L10C17
Background

" TRAF2 (TNF receptor-associated factor 2) is a key adaptor protein in the TRAF family that regulates signals from TNFR superfamily members, including TNF-α and CD40. TRAF2 phosphorylation at serine 11, mediated by kinases like TANK-binding kinase 1 (TBK1), is critical for the activation of downstream JNK and NF-κB signaling pathways. This phosphorylation promotes cytosolic translocation of TRAF2, interferes with its RING domain’s interaction with phospholipids, and is required for full activation of the JNK pathway and the secondary phase of the NF-κB pathway. Functionally, TRAF2 phosphorylation enhances c-Jun and NF-κB activities and confers resistance to stress-induced apoptosis, making it a potential therapeutic target in cancer, where altered TRAF2 signaling contributes to tumorigenesis and disease progression."
References

技術サポート

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