Cat. # | Size | Qty. | Price |
---|---|---|---|
12387S | 100 µl |
|
REACTIVITY | H M R |
SENSITIVITY | Endogenous |
MW (kDa) | 95 |
Source/Isotype | Rabbit IgG |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:50 |
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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 (#59329, 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 June 2020
Protocol Id: 10
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, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 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 2021
Protocol Id: 410
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val78 of human UHF1 protein.
Ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1), also known as Inverted CCAAT box-binding protein of 90 kDa (ICBP90) and Nuclear Zinc Finger Protein NP95 (NP95), is a nuclear protein that was first discovered as a CCAAT box-binding protein that regulates the expression of the Topoisomerase IIα (TOP2A) and Rb1 genes (1,2). Later studies have shown that UHRF1 is required for maintenance of CpG DNA methylation, the process of copying pre-existing methylation patterns onto the newly synthesized DNA strand after DNA replication (3-5). UHRF1 localizes primarily with highly methylated pericentromeric heterochromatin and is required for proper structure and function of these regions of the genome (6,7). However, UHRF1 also localizes to euchromatic regions of the genome and functions to negatively regulate the expression of a subset of tumor suppressor genes (2,8,9). The localization and repressive functions of UHRF1 are both mediated by several protein domains, including a ubiquitin-like (UBL) domain, Tudor domain, PHD domain, SET and RING finger-associated (SRA) domain, and a RING finger domain. The SRA domain of UHRF1 binds with high affinity to hemi-methylated DNA and functions to properly target the associated maintenance DNA methyltransferase DNMT1 protein to mediate faithful methylation of the newly synthesized DNA strand (3-5). Additional targeting of UHRF1 to heterochromatin is mediated by the Tudor domain, which binds specifically to tri-methylated Lys9 of histone H3, a histone mark associated with pericentromeric heterochromatin (10-12). Targeting of UHRF1 to euchromatin is further mediated by the PHD domain, which binds specifically to unmethylated Arg2 of histone H3, which is commonly associated with euchromatin (13). In addition to recruiting DNMT1, UHRF1 recruits the histone deacetylase 1 (HDAC1) protein to target loci, resulting in deacetylation of histones, and providing an additional mechanism for transcriptional repression (3). Taken together, these studies demonstrate that UHRF1 functions to link DNA methylation and histone modifications to the maintenance of repressive chromatin structures. These functions of UHRF1 are important for proper maintenance of cell growth and proliferation, as research studies have shown UHRF1 overexpression in a number of cancers (breast, lung, colon, and prostate cancer) is associated with increased proliferation and malignancy (9,14-16).
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