Protein A Antibody [B17N6]

Catalog No.: F4409

Application: Reactivity: Staphylococcus aureus

Lane 1: Recombinant Alkalitolerant Protein 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
IP1:400-1:800

Application
IP, IF, ELISA

Source
Mouse Monoclonal Antibody

Reactivity
Staphylococcus aureus

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
~42 kDa

ポジティブコントロール	Protein A from Staphylococcus aureus
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
Protein A Antibody [B17N6] detects endogenous levels of total Protein A protein.

Clone
B17N6

Background
Protein A (SpA) is a major surface virulence factor of Staphylococcus aureus and a member of the immunoglobulin-binding protein family. It is expressed on the bacterial cell wall with multiple tandem IgG-binding domains. SpA has high affinity for the Fc region of IgG from humans, rabbits, and other mammals, but a low affinity for mouse IgG1. This interaction allows SpA to interfere with host immune defenses by binding the Fcγ domain of IgG, blocking the Fab arms from opsonizing the bacterial surface, and preventing phagocytosis by neutrophils and macrophages. As a result, S. aureus can evade clearance in bloodstream infections. SpA also acts as a B cell superantigen by cross-linking B cell receptors that use VH3-family Fabs, leading to extensive polyclonal B cell expansion and subsequent apoptosis. This cripples adaptive immunity by deleting B-1 and marginal zone B cells, resulting in long-term defects in humoral immunity and promoting recurrent infections. The protein activates tyrosine kinase signaling pathways through phospholipase C, PKC, and MAP kinases, causing aberrant B cell proliferation before deletion. SpA is widely used for antibody purification by affinity chromatography and for detection in Western blots, radioimmunoassays (RIAs), and immunoprecipitations due to its conserved Fc binding. Monoclonal anti-SpA IgG1 antibodies are used to detect and quantify SpA-tagged fusion proteins and S. aureus bacteria. Dysregulated SpA expression contributes to chronic and methicillin-resistant S. aureus (MRSA) infections.

Background

Protein A (SpA) is a major surface virulence factor of Staphylococcus aureus and a member of the immunoglobulin-binding protein family. It is expressed on the bacterial cell wall with multiple tandem IgG-binding domains. SpA has high affinity for the Fc region of IgG from humans, rabbits, and other mammals, but a low affinity for mouse IgG1. This interaction allows SpA to interfere with host immune defenses by binding the Fcγ domain of IgG, blocking the Fab arms from opsonizing the bacterial surface, and preventing phagocytosis by neutrophils and macrophages. As a result, S. aureus can evade clearance in bloodstream infections. SpA also acts as a B cell superantigen by cross-linking B cell receptors that use VH3-family Fabs, leading to extensive polyclonal B cell expansion and subsequent apoptosis. This cripples adaptive immunity by deleting B-1 and marginal zone B cells, resulting in long-term defects in humoral immunity and promoting recurrent infections. The protein activates tyrosine kinase signaling pathways through phospholipase C, PKC, and MAP kinases, causing aberrant B cell proliferation before deletion. SpA is widely used for antibody purification by affinity chromatography and for detection in Western blots, radioimmunoassays (RIAs), and immunoprecipitations due to its conserved Fc binding. Monoclonal anti-SpA IgG1 antibodies are used to detect and quantify SpA-tagged fusion proteins and S. aureus bacteria. Dysregulated SpA expression contributes to chronic and methicillin-resistant S. aureus (MRSA) infections.

References
[1] Das T, et al. Immunol Lett. 1999 Oct 1;70(1):43-51. [2] Falugi F, et al. mBio. 2013 Aug 27;4(5):e00575-13.

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%