REACTIVITY | SENSITIVITY | MW (kDa) | Isotype |
---|---|---|---|
H | Endogenous | 30 | Rabbit IgG |
Western blot analysis of extracts from various cell lines using HUS1 (D4J9H) Rabbit mAb.
Learn more about how we get our imagesImmunoprecipitation of HUS1 from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb XP® Isotype Control #3900, and lane 3 is HUS1 (D4J9H) Rabbit mAb. Western blot analysis was performed using HUS1 (D4J9H) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.
Learn more about how we get our imagesFor 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 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 October 2017
Protocol Id: 410
Application | Dilutions |
---|---|
Western Blotting | 1:1000 |
Immunoprecipitation | 1:200 |
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.
HUS1 (D4J9H) Rabbit mAb recognizes endogenous levels of total HUS1 protein. In some cell lysates, this antibody detects a 45 kDa band of unknown origin.
Human
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gln147 of human HUS1 protein.
DNA damage resulting from genotoxic stress activates cellular checkpoints that prevent or delay cell division until damaged DNA is repaired or the cell follows an apoptotic pathway. The Rad9 homolog A (Rad9A, Rad9) protein is part of a checkpoint protein complex that acts as an early sensor of DNA damage. Together with the HUS1 and Rad1 checkpoint proteins, Rad9 forms a heterotrimeric 9-1-1 complex with a ring structure similar to the processivity factor PCNA. The 9-1-1 complex induces multiple signaling pathways, including the ATM- and ATR-activated DNA repair pathways (1,2). A functional 9-1-1 complex is required for ATR-dependent S phase checkpoint signaling (3).
The 9-1-1 complex interacts with DNA topoisomerase 2-binding protein 1 (TopBP1) in response to DNA damage, activating ATR and causing signal amplification through further recruitment of TopBP1 (4). The 9-1-1 complex interacts with DNA mismatch repair proteins MSH2, MSH3, and MSH6 to play a role in mismatch repair (5). During an error-free DNA damage tolerance process, the 9-1-1 complex cooperates with polyubiquitinated PCNA and Exo1 nuclease to support switching of the replicative polymerase to the undamaged template (6).
Cell Signaling Technology 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 |
---|---|---|
16416S | 100 µl (10 western blots) | $ 255.0 |