Rabbit IgG Isotype Control Antibody (Rabbit mAb) [D24D4]

Catalog No.: F2665

Application: Reactivity:

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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

Application
IP, IHC, IF, FCM, ChIP

Source
Rabbit Monoclonal Antibody

Reactivity

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	HeLa cells; A549 cells; Hep G2 cells
ネガティブコントロール	Human colon carcinoma

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.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

生物学的記述

Specificity
Rabbit IgG Isotype Control Antibody (Rabbit mAb) [D24D4] functions as an isotype control for rabbit IgG antibodies.

Clone
D24D4

Background
Rabbit IgG isotype control antibodies are immunoglobulin G class rabbit antibodies that possess the same constant region and Y-shaped structure as target-specific rabbit IgG, including paired heavy and light chains with an Fc region capable of engaging Fc receptors and complement, and Fab regions that lack specificity for antigens in the test system. The Fc portion retains the ability to bind Fcγ receptors, interact with Fc-binding proteins, and participate in non-antigen-driven associations with cellular lipids, carbohydrates, and extracellular matrix components, while the variable domains do not recognize the epitopes under investigation, resulting in a nonspecific binding profile equivalent to that of a typical rabbit primary antibody without contributing target-directed signal. Under experimental conditions, rabbit IgG isotype controls serve as negative controls to define background resulting from Fc receptor binding, weak protein–protein or protein–surface interactions, and fluorophore or enzyme conjugates, and are used at the same immunoglobulin class, subclass, and concentration as the test antibody to ensure equivalent avidity, Fc interactions, and labeling density. In flow cytometry, immunohistochemistry, and immunofluorescence, isotype control IgG binds Fc receptors on leukocytes and tissue-resident immune cells and engages nonspecific sites to the same extent as a typical rabbit primary antibody, allowing direct comparison of signal between isotype and test antibody channels and establishing a threshold that separates specific antigen-dependent staining from background. During chromatin immunoprecipitation, RIP, ELISA, or immunoprecipitation, normal rabbit IgG functions as a matrix-binding and chromatin-binding control that reports non-epitope-directed capture of nucleic acids and proteins on support surfaces under conditions identical to those used for the antigen-specific antibody. The constant-region structure of rabbit IgG enables recognition by anti-rabbit secondary antibodies and by protein A or protein G, allowing rabbit IgG isotype controls to integrate into detection and purification systems and report signal originating from secondary reagents, enzyme or fluorophore tags, or capture matrices. In multiparameter flow cytometry panels using directly conjugated rabbit antibodies, conjugated rabbit IgG isotype controls match fluorophore, isotype, and concentration, quantifying background from fluorochrome-related and Fc-mediated interactions and enabling gates and positivity thresholds to be set relative to this baseline. Across these applications, rabbit IgG isotype controls enhance assay robustness in samples with abundant Fc receptor–positive cells or high intrinsic autofluorescence by distinguishing true antigen-dependent signal from noise due to Fc–ligand interactions and nonspecific adsorption. In disease-oriented and translational studies reliant on antibody-based readouts, rabbit IgG isotype controls provide a reference for interpreting biomarker expression levels and distribution, ensuring that staining patterns attributed to pathology are not explained by altered Fc receptor expression or matrix composition alone.

Background

Rabbit IgG isotype control antibodies are immunoglobulin G class rabbit antibodies that possess the same constant region and Y-shaped structure as target-specific rabbit IgG, including paired heavy and light chains with an Fc region capable of engaging Fc receptors and complement, and Fab regions that lack specificity for antigens in the test system. The Fc portion retains the ability to bind Fcγ receptors, interact with Fc-binding proteins, and participate in non-antigen-driven associations with cellular lipids, carbohydrates, and extracellular matrix components, while the variable domains do not recognize the epitopes under investigation, resulting in a nonspecific binding profile equivalent to that of a typical rabbit primary antibody without contributing target-directed signal. Under experimental conditions, rabbit IgG isotype controls serve as negative controls to define background resulting from Fc receptor binding, weak protein–protein or protein–surface interactions, and fluorophore or enzyme conjugates, and are used at the same immunoglobulin class, subclass, and concentration as the test antibody to ensure equivalent avidity, Fc interactions, and labeling density. In flow cytometry, immunohistochemistry, and immunofluorescence, isotype control IgG binds Fc receptors on leukocytes and tissue-resident immune cells and engages nonspecific sites to the same extent as a typical rabbit primary antibody, allowing direct comparison of signal between isotype and test antibody channels and establishing a threshold that separates specific antigen-dependent staining from background. During chromatin immunoprecipitation, RIP, ELISA, or immunoprecipitation, normal rabbit IgG functions as a matrix-binding and chromatin-binding control that reports non-epitope-directed capture of nucleic acids and proteins on support surfaces under conditions identical to those used for the antigen-specific antibody. The constant-region structure of rabbit IgG enables recognition by anti-rabbit secondary antibodies and by protein A or protein G, allowing rabbit IgG isotype controls to integrate into detection and purification systems and report signal originating from secondary reagents, enzyme or fluorophore tags, or capture matrices. In multiparameter flow cytometry panels using directly conjugated rabbit antibodies, conjugated rabbit IgG isotype controls match fluorophore, isotype, and concentration, quantifying background from fluorochrome-related and Fc-mediated interactions and enabling gates and positivity thresholds to be set relative to this baseline. Across these applications, rabbit IgG isotype controls enhance assay robustness in samples with abundant Fc receptor–positive cells or high intrinsic autofluorescence by distinguishing true antigen-dependent signal from noise due to Fc–ligand interactions and nonspecific adsorption. In disease-oriented and translational studies reliant on antibody-based readouts, rabbit IgG isotype controls provide a reference for interpreting biomarker expression levels and distribution, ensuring that staining patterns attributed to pathology are not explained by altered Fc receptor expression or matrix composition alone.

References
[1] Weber J, et al. Exp Mol Med. 2017 Mar 24;49(3):e305. [2] Pleiner T, et al. J Cell Biol. 2018 Mar 5;217(3):1143-1154.

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%