Recombinant Anti-MBD3 antibody [EPR18258] KO Tested (ab188401)

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Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR18258] to MBD3
Suitable for: WB, ICC/IF, IHC-P
Knockout validated
Reacts with: Mouse, Rat, Human

Carrier Free

Product name

Anti-MBD3 antibody [EPR18258]
See all MBD3 primary antibodies
Description

Rabbit monoclonal [EPR18258] to MBD3
Host species

Rabbit
Specificity

This antibody detects a doublet by western blot, which we believe corresponds to isoforms 1 and 2 of MBD3.

This product is only recommended for human IHC but not for mouse or rat.
Tested applications

Suitable for: WB, ICC/IF, IHC-Pmore details
Species reactivity

Reacts with: Mouse, Rat, Human
Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: HeLa, 293, F9, WEHI-3, T-47D, HepG2, C6, RAW 264.7, PC-12 and NIH/3T3 whole cell lysates; Human fetal kidney lysate; Mouse spleen lysate. ICC/IF: HeLa and NIH/3T3 cells.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 0.05% BSA, 40% Glycerol (glycerin, glycerine)
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR18258
Isotype

IgG
Research areas

Alternative Versions
- Anti-MBD3 antibody [EPR18258] - BSA and Azide free (ab250961)
Compatible Secondaries
- Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
- Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
Recombinant Protein
- Recombinant Human MBD3 protein (ab134609)

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab188401 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
WB		1/2000. Detects a band of approximately 33, 29 kDa (predicted molecular weight: 33, 29 kDa).
ICC/IF		1/500.
IHC-P		1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Notes
WB 1/2000. Detects a band of approximately 33, 29 kDa (predicted molecular weight: 33, 29 kDa).
ICC/IF 1/500.
IHC-P 1/200. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Function

Does not bind DNA by itself. Recruits histone deacetylases and DNA methyltransferases. Acts as transcriptional repressor and plays a role in gene silencing.
Sequence similarities

Contains 1 MBD (methyl-CpG-binding) domain.
Cellular localization

Nucleus. Nuclear, in discrete foci.
Target information above from: UniProt accession O95983 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 53615 Human
- Entrez Gene: 17192 Mouse
- Entrez Gene: 362834 Rat
- Omim: 603573 Human
- SwissProt: O95983 Human
- SwissProt: Q9Z2D8 Mouse
- Unigene: 178728 Human
- Unigene: 7142 Mouse
Alternative names
- AI181826 antibody
- AU019209 antibody
- MBD 3 antibody
- Mbd3 antibody
- MBD3: methyl CpG binding domain protein 3 antibody
- MBD3_HUMAN antibody
- Methyl CpG binding domain protein 3 antibody
- Methyl CpG binding protein MBD3 antibody
- Methyl-CpG-binding domain protein 3 antibody
- Methyl-CpG-binding protein MBD3 antibody
see all

Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: MBD3 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: F9 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab188401 observed at 33 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab188401 was shown to specifically react with MBD3 when MBD3 knockout samples were used. Wild-type and MBD3 knockout samples were subjected to SDS-PAGE. ab188401 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/10000 and incubated respectively overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MBD3 antibody [EPR18258] (ab188401)

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling MBD3 with ab188401 at 1/200 dilution (6.17 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human stomach. The section was incubated with ab188401 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR18258] (ab188401)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling MBD3 with ab188401 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab188401 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.
Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)

All lanes : Anti-MBD3 antibody [EPR18258] (ab188401) at 1/5000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
Lane 2 : 293 (Human epithelial cells from embryonic kidney) whole cell lysate
Lane 3 : F9 (Mouse embyro testicular cancer cell line) whole cell lysate
Lane 4 : WEHI-3 (Mouse leukemia cell line) whole cell lysate
Lane 5 : T-47D (Human ductal breast epithelial tumor cell line) whole cell lysate
Lane 6 : HepG2 (Human liver hepatocellular carcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 33, 29 kDa
Observed band size: 29,33 kDa why is the actual band size different from the predicted?

Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.
The 33 and 29 kDa bands correspond to Isoforms 1 and 2.
Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR18258] (ab188401)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling MBD3 with ab188401 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab188401 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MBD3 antibody [EPR18258] (ab188401)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling MBD3 with ab188401 at 1/200 dilution (6.17 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human pancreas (PMID: 27898661). The section was incubated with ab188401 for 15 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)

All lanes : Anti-MBD3 antibody [EPR18258] (ab188401) at 1/2000 dilution

Lane 1 : C6 (Rat glial tumor cells) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 33, 29 kDa
Observed band size: 29,33 kDa why is the actual band size different from the predicted?

Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.
The 33 and 29 kDa bands correspond to Isoforms 1 and 2.
Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)

Anti-MBD3 antibody [EPR18258] (ab188401) at 1/2000 dilution + Human fetal kidney tissue lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 33, 29 kDa
Observed band size: 29,33 kDa why is the actual band size different from the predicted?

Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.
Western blot - Anti-MBD3 antibody [EPR18258] (ab188401)

Anti-MBD3 antibody [EPR18258] (ab188401) at 1/2000 dilution + Mouse spleen tissue lysate at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 33, 29 kDa
Observed band size: 29,33 kDa why is the actual band size different from the predicted?

Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.
The 33 and 29 kDa bands correspond to Isoforms 1 and 2.
Anti-MBD3 antibody [EPR18258] (ab188401)

Western blot protocols
Immunocytochemistry & immunofluorescence protocols

Click here to view the general protocols

SDS download

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Datasheet download

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Publishing research using ab188401? Please let us know so that we can cite the reference in this datasheet.

ab188401 has been referenced in 1 publication.

Zhan X et al. Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex-mediated STAT1 downregulation. J Exp Med 217:N/A (2020). PubMed: 32181805

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Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Specificity

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Isotype control

Recombinant Protein

Applications

The Abpromise guarantee

Target

Function

Sequence similarities

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

Certificate of Compliance

References (1)

Customer reviews and Q&As