14-3-3 (pan) Rabbit mAb

Catalog No.: F2658

Lane 1: A431
Lane 2: 3T3
Lane 3: COS-7

サイズ（液体）	価格(税別)	在庫状況
20uL	JPY 19800	国内在庫なし(納期7~10日)
100uL	JPY 49500	国内在庫なし(納期7~10日)
100uL*2	JPY 74200	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, 细胞核, 分泌/細胞外環境, 細胞内膜系。
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。
転写条件（ウェット）: 200 mA, 60 min。

使用情報

Dilution
WB1:1000

Application
WB

Source
Rabbit

Reactivity
Human, Mouse, Rat, 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
27-29 kDa 27 kDa-29 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	A549; NIH/3T3; H-4-II-E; COS-7
ネガティブコントロール

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)，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) 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) 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, 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 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)，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) 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) 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, 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 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
14-3-3 (pan) Rabbit mAb recognizes endogenous levels of total 14-3-3 protein. This antibody detects all known isoforms of mammalian 14-3-3 proteins (β/α, γ, ε, η, ζ/δ, θ/τ and σ).

Synonym(s)
pan 14-3-3

Uniprot ID
P62258 , P61981, P31946, P27348, Q04917, P31947, P63104

Clone
N16K10

Background
14-3-3 proteins are a family of ubiquitously expressed phosphoserine and phosphothreonine-binding proteins that play a crucial role in regulating cellular processes, including signal transduction, apoptosis, checkpoint control, and nutrient sensing. Humans have seven isoforms of 14-3-3 proteins, labeled β, γ, ε, η, σ, τ, and ζ. Each dimeric protomer, consisting of two 30 kDa subunits, contains nine α-helices and a phosphopeptide-binding site, forming a cup-shaped structure with a central channel. These proteins regulate the subcellular localization of various binding partners, such as promoting the cytoplasmic localization of pro-apoptotic proteins like BAD or facilitating the nuclear localization of telomerase. With seven isoforms in humans, 14-3-3 proteins are critical for maintaining cellular homeostasis and are implicated in diseases such as cancer, where specific isoforms are often upregulated. Their ability to modulate a wide range of pathways underscores their importance in cellular function and disease progression.

Background

14-3-3 proteins are a family of ubiquitously expressed phosphoserine and phosphothreonine-binding proteins that play a crucial role in regulating cellular processes, including signal transduction, apoptosis, checkpoint control, and nutrient sensing. Humans have seven isoforms of 14-3-3 proteins, labeled β, γ, ε, η, σ, τ, and ζ. Each dimeric protomer, consisting of two 30 kDa subunits, contains nine α-helices and a phosphopeptide-binding site, forming a cup-shaped structure with a central channel. These proteins regulate the subcellular localization of various binding partners, such as promoting the cytoplasmic localization of pro-apoptotic proteins like BAD or facilitating the nuclear localization of telomerase. With seven isoforms in humans, 14-3-3 proteins are critical for maintaining cellular homeostasis and are implicated in diseases such as cancer, where specific isoforms are often upregulated. Their ability to modulate a wide range of pathways underscores their importance in cellular function and disease progression.

References
[1] Obsilova V, et al. Front Mol Biosci. 2022 Sep 16;9:1016071.

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%