Influenza A Virus M2 Protein Antibody [L21L24]

Catalog No.: F2238

Application: Reactivity: Influenza A

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代表番号: 045-509-1970|電子メール：sales@selleck.co.jp

使用情報

Dilution
IF1:2000

Application
IF

Source
Mouse Monoclonal Antibody

Reactivity
Influenza A

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

ポジティブコントロール	Dog MDCK cell
ネガティブコントロール

プロトコール

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

生物学的記述

Specificity
Influenza A Virus M2 Protein Antibody [L21L24] detects exogenous levels of total Influenza A Virus M2 protein.

タンパク質の局在
宿主膜、細胞内膜系、宿主細胞膜、ビリオン

Uniprot ID
P05780; P36348

Clone
L21L24

Synonym(s)
Influenza A virus matrix protein M2; Matrix protein 2; Membrane ion channel M2; Membrane protein M2; Proton channel protein M2

Background
The Influenza A Virus (IAV) M2 protein is a 97-amino acid, integral membrane viroporin essential for the viral life cycle, functioning as a homotetrameric, pH-activated, proton-selective ion channel. Its structure consists of an N-terminal ectodomain (residues 1–24) critical for virion incorporation, a transmembrane domain (residues 25–43) forming the channel core, and a C-terminal domain (residues 44–97) that includes an amphipathic helix and an unstructured tail involved in virus assembly and budding. Key residues, His37 and Trp41, serve as the proton sensor and channel gate, respectively. Upon endosomal acidification during viral entry, M2 mediates proton influx into the virion, facilitating release of viral ribonucleoproteins for replication. Subsequently, M2 maintains optimal pH in the trans-Golgi network to prevent premature hemagglutinin activation, while its C-terminal amphipathic helix induces membrane curvature required for viral budding and scission. M2 activity is regulated by pH and host protein interactions, and although it is a target of antivirals like amantadine, drug resistance is common due to mutations in the transmembrane domain.

Background

The Influenza A Virus (IAV) M2 protein is a 97-amino acid, integral membrane viroporin essential for the viral life cycle, functioning as a homotetrameric, pH-activated, proton-selective ion channel. Its structure consists of an N-terminal ectodomain (residues 1–24) critical for virion incorporation, a transmembrane domain (residues 25–43) forming the channel core, and a C-terminal domain (residues 44–97) that includes an amphipathic helix and an unstructured tail involved in virus assembly and budding. Key residues, His37 and Trp41, serve as the proton sensor and channel gate, respectively. Upon endosomal acidification during viral entry, M2 mediates proton influx into the virion, facilitating release of viral ribonucleoproteins for replication. Subsequently, M2 maintains optimal pH in the trans-Golgi network to prevent premature hemagglutinin activation, while its C-terminal amphipathic helix induces membrane curvature required for viral budding and scission. M2 activity is regulated by pH and host protein interactions, and although it is a target of antivirals like amantadine, drug resistance is common due to mutations in the transmembrane domain.

References
[1] Cady SD, et al. Biochemistry. 2009 Aug 11;48(31):7356-64. [2] Ma C, et al. Proc Natl Acad Sci U S A. 2009 Jul 28;106(30):12283-8.

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%