HDAC7 Rabbit mAb

Catalog No.: F1252

Lane 1: COS-7
Lane 2: K-562
Lane 3: Hela
Lane 4: LNCap
Lane 5: NIH/3T3
Immunohistochemical analysis of formalin fixed paraffin embedded human Urothelial Carcinoma tissue with F1252 at 1/100 dilution.

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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

カスタマーフィードバック(1)

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：5%。

使用情報

Dilution
WB1:1000 IHC1:100

Application
WB, IHC

Source
Rabbit

Reactivity
Human, Mouse, 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
124 Kda 124 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	Human breast carcinoma/ovarian carcinoma/tonsil; K-562; HeLa; LNCaP; COS-7; NIH/3T3
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: 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 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: 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 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
HDAC7 Rabbit mAb recognizes endogenous levels of total HDAC7 protein. This antibody does not cross-react with other HDAC proteins, including HDAC4 and HDAC5.

Uniprot ID
Q8WUI4

Clone
M24H5

Background
HDAC7, a member of the histone deacetylase (HDAC) family, is essential for modifying histones and regulating gene expression. It catalyzes lysine deacetylation, leading to chromatin compaction and gene suppression. It plays a crucial role in inflammatory processes and immune response regulation, particularly in astrocyte-mediated inflammation linked to psychiatric disorders such as anxiety. During thymic selection, HDAC7 is critical in CD4+CD8+ double-positive thymocytes, inhibiting Nur77 expression to prevent apoptosis during negative selection. In macrophages, it acts as a metabolic switch, initiating tailored responses like glycolysis and proinflammatory reactions. However, it shifts to activating the pentose phosphate pathway (PPP) in the presence of nearby bacterial threats, empowering macrophages to adjust immune responses based on danger signals. Moreover, HDAC7's interaction with IKK proteins activates NF-κB signalling, enhancing proinflammatory gene expression. Dysregulation of HDAC7 affects tumor prognosis and various behaviors like proliferation, migration, apoptosis, and angiogenesis.

Background

HDAC7, a member of the histone deacetylase (HDAC) family, is essential for modifying histones and regulating gene expression. It catalyzes lysine deacetylation, leading to chromatin compaction and gene suppression. It plays a crucial role in inflammatory processes and immune response regulation, particularly in astrocyte-mediated inflammation linked to psychiatric disorders such as anxiety. During thymic selection, HDAC7 is critical in CD4+CD8+ double-positive thymocytes, inhibiting Nur77 expression to prevent apoptosis during negative selection. In macrophages, it acts as a metabolic switch, initiating tailored responses like glycolysis and proinflammatory reactions. However, it shifts to activating the pentose phosphate pathway (PPP) in the presence of nearby bacterial threats, empowering macrophages to adjust immune responses based on danger signals. Moreover, HDAC7's interaction with IKK proteins activates NF-κB signalling, enhancing proinflammatory gene expression. Dysregulation of HDAC7 affects tumor prognosis and various behaviors like proliferation, migration, apoptosis, and angiogenesis.

References
[1] Verdin E, et al. Novartis Found Symp. 2004;259:115-169. [2] Das Gupta K, et al. Proc Natl Acad Sci U S A. 2023;120(4):e2212813120.

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%