LAMTOR3/MAPKSP1 Rabbit mAb

Catalog No.: F1438

Lane 1: MCF7
Lane 2: mIMCD-3
Lane 3: Vero
Lane 4: AR42J
Lane 5: RD
Lane 6: COS-7
Immunohistochemical analysis of formalin fixed paraffin embedded human Colorectal cancer tissue with F1438 at 1/100 dilution.

サイズ（液体）	価格(税別)	在庫状況
50uL	JPY 23500	国内在庫なし(納期7~10日)
100uL	JPY 35500	国内在庫なし(納期7~10日)
100uL*2	JPY 53500	お問い合わせ

代表番号: 045-509-1970\|電子メール：[email protected] よく尋ねられる質問

キーポイント

タンパク質の局在：エンドソーム，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。
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
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
14 kDa

ポジティブコントロール	MCF-7; mIMCD-3; Vero; AR42J; RD; 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: 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 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: 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 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
LAMTOR3/MAPKSP1 Rabbit mAb recognizes endogenous levels of total LAMTOR3/MAPKSP1 protein. This antibody may cross-react with LAMTOR1/C11orf59 and LAMTOR2/ROBLD3.

Uniprot ID
Q9UHA4

Clone
L7A16

Background
LAMTOR3 (late endosomal/lysosomal adaptor and MAPK and MTOR activator 3), also known as MAPKSP1, is a roadblock domain-only subunit involved in tumorigenesis and development. It acts as a scaffold protein complex in the nucleus during anaphase mitosis and plays a critical role in various cancers by interacting with MAPK1/ERK2, a polymeric mitogen-activated protein kinase. LAMTOR3 promotes tumor cell growth and mitosis and is implicated in chemotherapy resistance in lung cancer by inducing BCL-2. Additionally, PCNA-associated factor (PAF) enhances pancreatic cancer cell proliferation via LAMTOR3 expression and MAPK pathway activation. LAMTOR3 also regulates apoptosis and holds potential as an important molecular marker. Amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. The Ragulator complex, encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation.

Background

LAMTOR3 (late endosomal/lysosomal adaptor and MAPK and MTOR activator 3), also known as MAPKSP1, is a roadblock domain-only subunit involved in tumorigenesis and development. It acts as a scaffold protein complex in the nucleus during anaphase mitosis and plays a critical role in various cancers by interacting with MAPK1/ERK2, a polymeric mitogen-activated protein kinase. LAMTOR3 promotes tumor cell growth and mitosis and is implicated in chemotherapy resistance in lung cancer by inducing BCL-2. Additionally, PCNA-associated factor (PAF) enhances pancreatic cancer cell proliferation via LAMTOR3 expression and MAPK pathway activation. LAMTOR3 also regulates apoptosis and holds potential as an important molecular marker. Amino acids induce the movement of mTORC1 to lysosomal membranes, where the Rag proteins reside. The Ragulator complex, encoded by the MAPKSP1, ROBLD3, and c11orf59 genes, interacts with the Rag GTPases, recruits them to lysosomes, and is essential for mTORC1 activation.

References
[1] Gong Y, et al. J Clin Lab Anal. 2022 Sep;36(9):e24648. [2] Sancak Y, et al. Cell. 2010 Apr 16;141(2):290-303.

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

技術サポート

ストックの作り方、阻害剤の保管方法、細胞実験や動物実験の際に注意すべき点など、製品を取扱う時に問い合わせが多かった質問に対しては取扱説明書でお答えしています。

Handling Instructions

他に質問がある場合は、お気軽にお問い合わせください。

* 必須

* 大学・企業名 大学・企業名を記入してください

* 名前 名前を記入してください

* 電子メール 電子メール・アドレスを記入してください 有効なメールアドレスを入力してください

電話番号

* 内容 お問い合わせ内容をご入力ください

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%