REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H M | Endogenous | 250, 280 | Rabbit IgG |
Western blot analysis of extracts from various cell lines using ARID1B/BAF250B (E1U7D) Rabbit mAb (upper) and α-Actinin (D6F6) Rabbit mAb #6487 (lower). MIA PaCa-2 cells do not express ARID1B/BAF250B protein.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded SH-SY5Y cell pellet (left, positive) or MIA PaCa-2 cell pellet (right, negative) using ARID1B/BAF250B (E1U7D) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using ARID1B/BAF250B (E1U7D) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human colon carcinoma using ARID1B/BAF250B (E1U7D) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using ARID1B/BAF250B (E1U7D) Rabbit mAb.
Learn more about how we get our images.Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using ARID1B/BAF250B (E1U7D) Rabbit mAb.
Learn more about how we get our images.For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Loading of prestained molecular weight markers (#13953, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.
IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:
Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.
IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.
IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2018
Protocol Id: 410
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted February 2010
revised March 2016
Protocol Id: 283
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:100 |
Immunohistochemistry (Paraffin) | 1:500 |
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
ARID1B/BAF250B (E1U7D) Rabbit mAb recognizes endogenous levels of total ARID1B/BAF250B protein. This antibody does not cross-react with ARID1A/BAF250A protein.
Human, Mouse
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala1320 of human ARID1B/BAF250B protein.
ATP-dependent chromatin remodeling complexes play an essential role in the regulation of nuclear processes such as transcription and DNA replication and repair (1,2). The SWI/SNF chromatin remodeling complex consists of more than 10 subunits and contains a single molecule of either BRM or BRG1 as the ATPase catalytic subunit. The activity of the ATPase subunit disrupts histone-DNA contacts and changes the accessibility of crucial regulatory elements to the chromatin. The additional core and accessory subunits play a scaffolding role to maintain stability and provide surfaces for interaction with various transcription factors and chromatin (2-5). The interactions between SWI/SNF subunits and transcription factors, such as nuclear receptors, p53, Rb, BRCA1, and MyoD, facilitate recruitment of the complex to target genes for regulation of gene activation, cell growth, cell cycle, and differentiation processes (1,6-9).
ARID1B (A-T rich interacting domain 1B), also known as BAF250B, is a DNA-binding member of the SWI/SNF complex. It has 60% homology with ARID1A/BAF250A, and the proteins are mutually exclusive members of the complex, akin to Brg1 and BRM (10). ARID1B plays a role in synapse formation and dendritic arborization in neuronal development, and haploinsufficiency of ARID1B has been reported in intellectual disability (11-13). Mutations in ARID1B have also been shown in Coffin-Siris syndrome (14). ARID1B/BAF250B is a critical vulnerability in ARID1A/BAF250A mutant cancers, and could be explored as a potential therapeutic target (15).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. SignalStain is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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Product # | Size | Price |
---|---|---|
65747S | 100 µl | N/A |
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