Anti-GRWD1 antibody, rabbit polyclonal, ChIP grade

Product#: 70-130
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Anti-GRWD1 antibody, rabbit polyclonal, ChIP grade


General information 

Cat. No. 70-130
Size 100 µg
Host Species Rabbit
Immunogen Purified GST-GRWD1 (human, full-length) expressed in E. coli.
Validation Validated for Western Blotting by siRNA.
KEYWORDS nucleolar stress response, oncogene, p53, PICT1, RPL11, tumor suppressor
Form 1 mg/ml in PBS and 50% glycerol. Filter-sterilized. Carrier protein and azide free.
Reactivity Human, mouse and rat. Other species have not been tested.
Purification The antiserum was first absorbed with GST-agarose column and then the pass-through fraction was affinity-purified with GST-GRWD1 agarose column.
Application :1. Western blotting (1/1,000~1/3,000 dilution)
2. Immunoprecipitation (Assay dependent)
3. Chromatin Immuno-Precipitation (Assay dependent)
4. Immunofluorescence staining / Immunochemistry (1/100~1/1,000 dilution)
Storage Ship at 4℃and store at -20℃. Do not freeze
Data Link uniprot/Q9BQ67 (GRWD1_HUMAN), uniprot/Q810D6 (GRWD1_MOUSE)

Description

Glutamate-rich WD repeat-containing protein 1 (GRWD1) consists of 446 amino acids with molecular mass of 49.4 kDa. It has been found as a protein which interacts with METTL18 and CDT1 proteins. It has been implicated in regulation of DNA replication and/or in ribosome biogenesis.
 
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Fig.1. Identication of GRWD1 proteins in whole cell lysates by western blotting with anti-GRWD1 antibody 

Whole cell lysates of HeLa cells untreated (-) and treated (+) with DNA damaging agent, adriamycin (Ad), and NIH3T3 cells were analyzed by western blotting with anti-GRWD1 antibody at 1/1,000 dilution. The samples were 10 μg. Second antibody was HRP-conjugated goat anti-rabbit IgG used at 1/5,000 dilution. The revelation of multiple bands indicates post-translational modification such as phosphorylation. The level of GRWD1 in the cell was not affected by DNA-damaging treatment. The identity of an additional band at ~85 kDa position other than the GRWD1 band in NIH-3T3 cell lysate is not known. The GRWD1 proteins were identified at a position higher (~55 kDa) than expected from the molecular mass of GRWD1 indicated from cDNA sequence (49.4 kDa).
 
Anti-GRWD1-2.png
Fig.2. Immunofluorescence staining of GRWD1 protein in HeLa cells.

Hela cells were fixed in 4% paraformaldehyde overnight and permeabilized in 0.25% TritonX 100 in PBS for 10 min. Anti-GRWD1 antibody was used at 1/1,000 dilution. As second antibody, goat anti-rabbit IgG conjugated with Alex488 was used at 1/5,000 dilution. As a signal enhancer, Can Get Signal Immunostain B (Toyobo, Osaka) was used according to the protocol of the supplier. Nuclei were stained with DAPI. GRWD1 protein is localized in nuclei.
Anti-GRWD1-3.png
Fig.3. Nucleolar localization of GRWD1.

HCT116 cells were first extracted with Triton X-100 to remove nucleoplasmic proteins, double-immunostained with anti-GRWD1 (green) and anti-fibrillarin (red) antibodies as a marker for nucleoli, and counterstained with DAPI. The anti-GRWD antibody was used at 1/500 dilution and as the second antibody, goat anti-rabbit IgG conjugated with Alex488 was used at 1/2,000 dilution.

 

References
This product has been described in Ref.1 and used in the following publication.

  1. Sugimoto N. et al. Identification of novel human Cdt1-binding proteins by a proteomics approach: proteolytic regulation by APC/CCdh1. Mol Biol Cell. 2008 19(3):1007-21. WB 
  2. Sugimoto N. et al. Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture. Nucleic Acids Res. 2015 Jul 13;43(12):5898-911. PMID:25990725 WB, IP, ChIP, IC/IF (human)
  3. Aizawa M. et al. Nucleosome assembly and disassembly activity of GRWD1, a novel Cdt1-binding protein that promotes pre-replication complex formation. Biochim Biophys Acta. 2016 Nov;1863(11):2739-2748. PMID:27552915 WB (human)
  4. Kayama K. et al. GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis. EMBO Rep. 2017 Jan;18(1):123-137. PMID:27856536 WB,IF, IP (human) 


Aliases for GRWD1 Gene

  • Glutamate Rich WD Repeat Containing 1 2 3 5
  • WDR28 2 3 4
  • GRWD 2 3 4
  • Glutamate-Rich WD Repeat-Containing Protein 1 3 4
  • RRB1 2 3
  • Regulator Of Ribosome Biogenesis 1 Homolog 3
  • CUL4- And DDB1-Associated WDR Protein 4 3
  • Glutamate Rich WD Repeat Protein GRWD 3
  • WD Repeat Domain 28 3
  • KIAA1942 4
  • GRWD1 5
  • CDW4 3
 
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