Anti-RYBP antibody [EPR13059(2)] - ChIP Grade

13 product images

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  ChIP - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Chromatin was prepared from NIH/3T3 cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
  The ChIP was performed with 25 µg of chromatin, 5 µg of ab185971 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
  Primers and probes are located in the first kb of the transcribed region.
  *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

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  Immunoprecipitation - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  ab185971 (purified) at 1/20 dilution (11.7μg/mL) immunoprecipitating RYBP in NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10 μg.
  
  Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate 10 μg
  Lane 2 (+): ab185971 & NIH/3T3 whole cell lysate
  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab185971 in NIH/3T3 whole cell lysate
  
  For western blotting, ab185971 at 1/500 dilution (0.468 μg/mL) and veriBlot for IP secondary antibody (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution was used.
  
  Blocking and diluting buffer: 5% NFDM /TBST.

  All lanes: Immunoprecipitation - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Predicted band size: 25 kDa

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  Western blot - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  All lanes: Western blot - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971) at 1/1000 dilution

  Lane 1: K562 cell lysate at 20 µg

  Lane 2: SW480 cell lysate at 20 µg

  Secondary

  All lanes: goat anti-rabbit IgG, (H+L), peroxidase conjugated at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 25 kDa

  Observed band size: 32 kDa

• 

  ChIP - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Chromatin was prepared from HEK293 cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
  The ChIP was performed with 25 µg of chromatin, 5 µg of ab185971 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
  Primers and probes are located in the first kb of the transcribed region.
  *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Immunohistochemical analysis of paraffin-embedded, Human clear cell carcinoma kidney tissue labeling RYBP with ab185971 at a 1/100 dilution. Counter stained with hematoxylin.
  

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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  Immunocytochemistry/ Immunofluorescence - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Immunofluorescence analysis of, paraformaldehyde-fixed, HepG2 cells labeling RYBP with ab185971 at a 1/250 dilution. As secondary antibody goat anti-rabbit IgG (Alexa Fluor®555) was used at a 1/200. In blue DAPI staining.

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  Flow Cytometry (Intracellular) - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Intracellular Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling RYBP with purified ab185971 at 1/20 dilution (11.7μg/mL) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (black). Unlabeled control - Unlabelled cells (blue).
  

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  Flow Cytometry (Intracellular) - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Intracellular Flow Cytometry analysis of paraformaldehyde-fixed K562 cells labeling RYBP with ab185971 at a 1/40 dilution and secondary antibody goat anti-rabbit IgG (FITC, red) at a 1/150 dilution, or negative control rabbit IgG (green).
  

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  Western blot - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  All lanes: Western blot - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971) at 1/10000 dilution

  All lanes: HepG2 cell lysate at 20 µg

  Secondary

  All lanes: goat anti-rabbit IgG, (H+L), peroxidase conjugated at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 25 kDa

  Observed band size: 32 kDa

• 

  Immunoprecipitation - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Western blot analysis on immunoprecipitation pellet from HepG2 cell lysate labeling RYBP with ab185971 at 1/50 dilution and goat anti-rabbit IgG, (H+L), peroxidase conjugated at a 1/1000 dilution.

  All lanes: Immunoprecipitation - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Predicted band size: 25 kDa

  Observed band size: 32 kDa

• 

  Western blot - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  All lanes: Western blot - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971) at 1/1000 dilution

  All lanes: rat PC12 cell lysate at 10 µg

  Secondary

  All lanes: goat anti-rabbit IgG, (H+L), peroxidase conjugated at 1/1000 dilution

  Developed using the ECL technique.

  Predicted band size: 25 kDa

  Observed band size: 32 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Immunohistochemical analysis of paraffin-embedded, Human placenta tissue labeling RYBP with ab185971 at a 1/100 dilution. Counter stained with hematoxylin.
  

  Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-RYBP antibody [EPR13059(2)] - ChIP Grade (ab185971)

  Immunofluorescence analysis of, paraformaldehyde-fixed, SW480 cells labeling RYBP with ab185971 at a 1/250 dilution. As secondary antibody goat anti-rabbit IgG (Alexa Fluor®555) was used at a 1/200. In blue DAPI staining.