Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade

40 product images

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  ChIP - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab176842 (red), and 20μl of Protein A/G sepharose beads. No antibody was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 with ab176842 at 1/2000 dilution, followed by Goat anti-rabbit Alexa Fluor® 488 (IgG) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (goat anti-mouse AlexaFluor®594 Goat secondary antibody) at 1/500 dilution (red).
  The negative controls are as follows;
  -ve control 1: ab176842 at 1/2000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
  -ve control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab176842 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) at 1 µg

  Lane 1: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 2: NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

  Lane 3: Drosophila embryo nuclear extract (from melanogaster embryos 0-12hr) at 10 µg

  Lane 4: S.cerevisiae (Y190) Whole Cell Lysate at 20 µg

  Lane 5: S.pombe Whole Cell Lysate at 20 µg

  Secondary

  All lanes: Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 15 kDa

  Observed band size: 17 kDa

  Exposure time: 5s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H3 with ab176842 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG; H&L) secondary antibody (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Nuclear staining on glandular epithelium of Human colon tissue is observed. Counter stained with Hematoxylin.
  

  Negative control: Used PBS instead of primary antibody, secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling Histone H3 with ab176842 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) secondary antibody (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Nuclear staining on mouse liver tissue is observed. Counter stained with Hematoxylin.
  

  Negative control: Used PBS instead of primary antibody, secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Immunohistochemical analysis of paraffin-embedded Rat pancreas tissue labeling Histone H3 with ab176842 at 1/500 dilution, followed by Goat Anti-Rabbit HRP (IgG H&L) secondary antibody (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution. Nuclear staining on rat pancreas tissue is observed. Counter stained with Hematoxylin.
  

  Negative control: Used PBS instead of primary antibody, secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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  Flow Cytometry (Intracellular) - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Histone H3 with purified ab176842 at 1/40 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.
  

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  Peptide Array - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  ab176842 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
  Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
  The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, is available under the Support & downloads section.

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST

  Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) was used as loading control at a 1/100000 dilution.

  Anti-6X His tag^® antibody [EPR20547] - ChIP Grade (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) was used as total protein control at a 1/5000 dilution.

  All lanes: Western blot - Anti-HIV1 p24 antibody [24-2] (Anti-HIV1 p24 antibody [24-2] ab85536) at 1/1000 dilution

  Lane 1: 293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a myc-His-tag® whole cell lysate at 4 µg

  Lane 2: 293T cells transfected with a Human immunodeficiency virus type 1 group M subtype B (isolate HXB2) (HIV-1) expression vector containing a myc-His-tag® whole cell lysate at 4 µg

  Lane 3: 293T cells transfected with a Human immunodeficiency virus type 1 group M subtype D (isolate NDK) (HIV-1) expression vector containing a myc-His-tag® whole cell lysate at 4 µg

  Lane 4: 293T cells transfected with a Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22) (HIV-1) expression vector containing a myc-His-tag® whole cell lysate at 4 µg

  Lane 5: 293T cells transfected with a Human immunodeficiency virus type 1 group M subtype K (isolate 96CM-MP535) (HIV-1) expression vector containing a myc-His-tag® whole cell lysate at 4 µg

  Secondary

  All lanes: Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

  Observed band size: 28 kDa

  Exposure time: 3s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Loading control: Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1:100000 (15Kda).

  All lanes: Western blot - Anti-Ref2P antibody [EPR28159-511] (Anti-Ref2P antibody [EPR28159-511] ab323859) at 1/1000 dilution

  Lane 1: SL2 (Drosophila melanogaster embryo epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

  Lane 2: SL2 transfected with siRNA specifically targeting Ref2P whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 110 kDa, 15 kDa

  Exposure time: 180s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 31559013).

  The identity of the higher MW band at approximately 70 kDa is unknown.

  In Western blot, Anti-H3 antibody (ab176842) staining at 1/50000 dilution.

  All lanes: Western blot - Anti-VIP antibody [EPR28121-6] (Anti-VIP antibody [EPR28121-6] ab324196) at 1/1000 dilution

  Lane 1: SK-N-SH (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: Capan-1 (human pancreatic adenocarcinoma epithelial cell) cytoplasmic fraction at 20 µg

  Lane 3: Capan-1 nuclear fraction at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 23 kDa, 24 kDa, 36 kDa, 15 kDa

  Exposure time: 6s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Lane 1: HaCaT (human skin keratinocyte) whole cell lysate

  Lane 2: HCC1937 (human mammary gland epithelial cell) whole cell lysate at 20 µg

  Observed band size: 93 kDa, 63 kDa

  Exposure time: 180s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  In Western blot, Anti-6X His tag antibody [EPR20547]-CHIP Grade (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.

  All lanes: Western blot - HRP Anti-V5 tag antibody [EPR27187-61] (HRP Anti-V5 tag antibody [EPR27187-61] ab323656) at 1/1000 dilution

  Lane 1: 293T cells transfected with an empty vector containing a His tag whole cell lysate at 20 µg

  Lane 2: 293T cells transfected with a V5 tag-Chromobox protein homolog 1 expression vector containing a His-tag whole cell lysate at 10 µg

  Lane 3: His-tagged human Chromobox protein homolog 1 recombinant protein at 50 ng

  Observed band size: 27 kDa, 15 kDa

  Exposure time: 3s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Low expression: AsPC-1.

  Alternative splicing results in multiple transcript variants.

  The identity of the bands between 20 kDa and 37 kDa are unknown.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - HRP Anti-GAA antibody [EPR4716(2)] (HRP Anti-GAA antibody [EPR4716(2)] ab323733) at 1/1000 dilution

  Lane 1: HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

  Lane 2: AsPC-1 (human Pancreas epithelial cell) whole cell lysate at 20 µg

  Lane 3: Human hippocampus tissue lysate at 20 µg

  Observed band size: 70-110 kDa, 15 kDa

  Exposure time: 3s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

  Alternatively spliced forms of the EVI-1 gene encode at least three distinct proteins: EVI-1 (145 kDa), MDS1/EVI-1 (200 kDa) and EVI-1Δ324 (88 kDa) PMID:(PMID: 24944602).

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-EVI1 antibody [EPR26816-18] (Anti-EVI1 antibody [EPR26816-18] ab315973) at 1/1000 dilution

  Lane 1: NIH/3T3 (mouse embryonic fibroblast) nuclear fraction at 20 µg

  Lane 2: NIH/3T3 cytoplasmic fraction at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 145 kDa, 200 kDa, 36 kDa, 15 kDa

  Exposure time: 169s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) (1:100000) (15KDa) staining at 1/1000000 dilution.

  The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and MMP7-KO homozygous mice (Strain ID: T012111).

  All lanes: Western blot - Anti-MMP7 antibody [EPR26105-26] (Anti-MMP7 antibody [EPR26105-26] ab302893) at 1/1000 dilution

  Lane 1: Wild-type mouse small intestine tissue lysate (male) at 40 µg

  Lane 2: MMP7 knockout mouse small intestine tissue lysate (male) at 40 µg

  Lane 3: Wild-type mouse small intestine tissue lysate (female) at 40 µg

  Lane 4: MMP7 knockout mouse small intestine tissue lysate (female) at 40 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 20 kDa

  Exposure time: 180s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Low expression: pancreas.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-Aldolase C antibody [EPR26896-61] - C-terminal (Anti-Aldolase C antibody [EPR26896-61] - C-terminal ab323506) at 1/1000 dilution

  Lane 1: Mouse cerebellum tissue lysate at 20 µg with NFDM/TBST

  Lane 2: Mouse brain tissue lysate at 20 µg with NFDM/TBST

  Lane 3: Mouse pancreas tissue lysate at 20 µg with NFDM/TBST

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Observed band size: 39 kDa, 15 kDa

  Exposure time: 1s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Negative control: SH-SY5Y (PMID:30650643 and PMID: 27345400)

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-Cytokeratin 19 antibody [EP1580Y] - Mouse IgG2a (Chimeric) (Anti-Cytokeratin 19 antibody [EP1580Y] - Mouse IgG2a (Chimeric) ab323562) at 1/1000 dilution

  Lane 1: MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 40 µg

  Secondary

  All lanes: Western blot - Goat Anti-Mouse IgG Fc (HRP) preadsorbed (Goat Anti-Mouse IgG Fc (HRP) preadsorbed ab98717) at 1/20000 dilution

  Observed band size: 44 kDa, 17 kDa

  Exposure time: 4s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  The identity of the higher MW band at approximately 50 kDa (in lane 2) is unknown.

  Samples are non-boiled as boiling may cause protein aggregation.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  In Western blot, Anti-Integrin alpha V antibody [EPR16800] (Anti-Integrin alpha V antibody [EPR16800] ab179475) staining at 1/5000 dilution.

  All lanes: Western blot - Anti-GPCR GPRC5D antibody [EPR28376-41] (Anti-GPCR GPRC5D antibody [EPR28376-41] ab315808) at 1/1000 dilution

  Lane 1: RPMI-8226 (human plasmacytoma, myeloma B lymphocyte) membrane fraction at 50 µg

  Lane 2: RPMI-8226 nuclear fraction at 50 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 33 kDa, 125 kDa, 15 kDa

  Exposure time: 15s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Low expression: pancreas, colon.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-Aldolase C antibody [EPR26896-61] - C-terminal (Anti-Aldolase C antibody [EPR26896-61] - C-terminal ab323506) at 1/1000 dilution

  Lane 1: Human cerebellum tissue lysate at 20 µg with NFDM/TBST

  Lane 2: Human pancreas tissue lysate at 20 µg with NFDM/TBST

  Lane 3: Human colon tissue lysate at 20 µg with NFDM/TBST

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Observed band size: 39 kDa, 15 kDa

  Exposure time: 1s

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  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  In Western blot, Anti-KMT2A / MLL + KMT2B / MLL4 antibody [EPR28282-16] - C-terminal ab315464 was shown to bind specifically to KMT2A. A band was observed at 180 kDa in wild-type HAP1 cell lysates whereas no signal observed at this size in KMT2A knockout cell line.

  This antibody has cross reaction with human KMT2B, band at 80 kDa represents KMT2B C-terminal protein.

  Lysates were freshly made and used immediately to minimize protein degradation.

  There is an unknown band at ~20 kDa.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-KMT2A / MLL + KMT2B / MLL4 antibody [EPR28282-16] - C-terminal (Anti-KMT2A / MLL + KMT2B / MLL4 antibody [EPR28282-16] - C-terminal ab315464) at 1/1000 dilution

  Lane 1: Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg with 5% NFDM/TBST

  Lane 2: KMT2A knockout HAP1 whole cell lysate at 20 µg with 5% NFDM/TBST

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Performed under reducing conditions.

  Observed band size: 180 kDa, 80 kDa, 15 kDa

  Exposure time: 81s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  
  Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as GAPDH loading control.
  
  ab176842 was for Histone H3 detection.

  All lanes: Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade ab32129) at 1/10000 dilution

  Lane 1: C6 (Rat glial tumor glial cell) whole cell lysate at 15 µg

  Lane 2: C6 (Rat glial tumor glial cell) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysate at 15 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  
  Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as GAPDH loading control.
  
  ab176842 was for Histone H3 detection.

  All lanes: Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade ab32129) at 1/10000 dilution

  Lane 1: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 15 µg

  Lane 2: NIH/3T3 (Mouse embryonic fibroblast) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysate at 15 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  
  Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was used as GAPDH loading control.
  
  ab176842 was for Histone H3 detection.

  All lanes: Western blot - Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade (Anti-Histone H3 (acetyl K9) antibody [Y28] - ChIP Grade ab32129) at 1/10000 dilution

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

  Lane 2: HeLa (Human cervix adenocarcinoma epithelial cell) treated with 500ng/ml Trichostatin A for 4 hours whole cell lysate at 15 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  In Western blot, Anti-Caspase-3 antibody [RM1299] ab323369 was shown to bind specifically to Caspase-3. Target of interest was observed at 32 kDa in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in Caspase-3 knockout cell line (lane 2).

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-Caspase-3 antibody [RM1299] (Anti-Caspase-3 antibody [RM1299] ab323369) at 1/1000 dilution

  Lane 1: Parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

  Lane 2: Caspase-3 knockout HAP1 whole cell lysate at 20 µg

  Lane 3: HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

  Lane 5: 293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

  Lane 6: Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

  Lane 7: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 32 kDa, 15 kDa

  Exposure time: 26s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-DAP12 antibody [EPR29151-41] (Anti-DAP12 antibody [EPR29151-41] ab323318) at 1/1000 dilution

  Lane 1: Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

  Lane 2: THP-1 cells differentiated with TPA (12-O-Tetradecanoylphorbol-13-acetate) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 11 kDa, 12 kDa, 15 kDa

  Exposure time: 26s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  Negative control: Jurkat (PMID: 8691135, 11841448), HEK-293.

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 8691135).

  In lanes 1-5, the lysates were stored at -80℃ prior to Western Blotting. The bands beneath the target band (46 kDa) are likely to be degradation products. In lanes 6-7, the lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  In Western blot, anti- H3 antibody (ab176842) loading control staining at 1/100000 dilution.

  Exposure time:

  Lanes 1-5: 48 seconds

  Lanes 6-7: 26 seconds

  All lanes: Western blot - Anti-SPIB antibody [EPR26784-117] (Anti-SPIB antibody [EPR26784-117] ab309346) at 1/1000 dilution

  Lane 1: Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

  Lane 2: HDLM-2 (human Hodgkin lymphoma) whole cell lysate at 20 µg

  Lane 3: Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

  Lane 4: Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

  Lane 5: HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

  Lane 6: Raji whole cell lysate at 20 µg

  Lane 7: Jurkat whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Observed band size: 46 kDa

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  In Western blot, anti- H3 antibody (ab176842) loading control staining at 1/100000 dilution.

  In Western blot, anti-GAPDH antibody (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/200000 dilution.

  All lanes: Western blot - Anti-EZHIP antibody [EPR28396-50] (Anti-EZHIP antibody [EPR28396-50] ab313392) at 1/1000 dilution

  Lane 1: Mouse testis cytoplasmic fraction at 50 µg

  Lane 2: Mouse testis nuclear fraction at 50 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Developed using the ECL technique.

  Observed band size: 75 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Low expression tissue: spleen, pancreas

  The identity of the lower MW band at approximately 100 kDa (in lane 1) is unknown.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution (15 kDa).

  All lanes: Western blot - Anti-nNOS (neuronal) antibody [RM1237] (Anti-nNOS (neuronal) antibody [RM1237] ab323183) at 1/1000 dilution

  Lane 1: Human hippocampus tissue lysate at 20 µg

  Lane 2: Human spleen tissue lysate at 20 µg

  Lane 3: Human pancreas tissue lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 161 kDa, 15 kDa

  Exposure time: 37s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  

  Negative control: kidney (PMID: 11922625).

  In Western blot, anti- H3 antibody (ab176842) loading control staining at 1/100000 dilution.

  This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

  All lanes: Western blot - Anti-DDX53/CT26 antibody [EPR27384-107] (Anti-DDX53/CT26 antibody [EPR27384-107] ab313333) at 1/1000 dilution

  Lane 1: Human testis tissue nuclear fraction at 40 µg

  Lane 2: Human kidney tissue nuclear fraction at 40 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Developed using the ECL technique.

  Observed band size: 74 kDa

  Exposure time: 70s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Low expression: Calu-3.

  The identity of the bands higher than 50 kDa are unknown.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-Aldolase C antibody [EPR26896-61] - C-terminal (Anti-Aldolase C antibody [EPR26896-61] - C-terminal ab323506) at 1/1000 dilution

  Lane 1: U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

  Lane 2: LNCaP (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

  Lane 3: Calu-3 (human lung adenocarcinoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

  Lane 4: RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg with NFDM/TBST

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 39 kDa, 15 kDa

  Exposure time: 8s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  The expression of phosphorylated FAK is upregulated in response to pervanadate treatment (PMID: 20847951).

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  In Western blot, Anti-FAK antibody [EP695Y] (Anti-FAK antibody [EP695Y] ab40794) staining at 1/1000 dilution.

  All lanes: Western blot - Anti-FAK (phospho Y397 + Y576 + Y925) antibody [RM1141] (Anti-FAK (phospho Y397 + Y576 + Y925) antibody [RM1141] ab322354) at 1/1000 dilution

  Lane 1: Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (untreated membrane) at 20 µg

  Lane 2: NIH/3T3 treated with 10μM pervanadate for 60 minutes whole cell lysate (untreated membrane) at 20 µg

  Lane 3: Untreated NIH/3T3 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

  Lane 4: NIH/3T3 treated with 10μM pervanadate for 60 minutes whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 119 kDa, 15 kDa

  Exposure time: 8s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Low expression: K-562 (PMID: 25960936), IMR-32, JAR, MCF7.

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 24173243).

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  Exposure time: Lanes 1-2: 1 second, lanes 3-7: 3 seconds

  All lanes: Western blot - Anti-HLA Class 1 ABCG antibody [EPR29051-62] (Anti-HLA Class 1 ABCG antibody [EPR29051-62] ab323616) at 1/1000 dilution

  Lane 1: KARPAS-299 (human T cell lymphoma cell) whole cell lysate at 20 µg

  Lane 2: A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 3: T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate at 20 µg

  Lane 4: K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

  Lane 5: IMR-32 (human neuroblastoma neuroblast) whole cell lysate at 20 µg

  Lane 6: JAR (human placenta choriocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 7: MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 40, 41 kDa, 15 kDa

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Histone H3 Western blot staining using rabbit Anti-Histone H3 antibody

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  This antibody does not cross-react with human PER1 and PER3.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1:100000 (15Kda); Anti-6X His tag® antibody [EPR20547] - ChIP Grade (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.

  All lanes: Western blot - Anti-PER2 antibody [EPR28181-56] (Anti-PER2 antibody [EPR28181-56] ab324161) at 1/1000 dilution

  Lane 1: 293T cells transfected with an empty vector containing a His-tag, whole cell lysate at 20 µg

  Lane 2: 293T cells transfected with a human PER2 expression vector containing a His-tag, whole cell lysate at 20 µg

  Lane 3: 293T cells transfected with a human PER1 expression vector containing a His-tag, whole cell lysate at 20 µg

  Lane 4: 293T cells transfected with a human PER3 expression vector containing a His-tag, whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 200 kDa, 20-250 kDa, 15 kDa

  Exposure time: 1s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Negative control: Neuro-2a, B16-F10 (PMID: 25858093).

  The molecular weight observed is consistent with what has been described in the literature (PMID: 38514849). The ~38 kDa band likely represents a N-terminal cleavage product of RIP3.

  This antibody was tested on frozen and fresh L-929, Neuro-2a and J774A.1 whole cell lysates and it was concluded that RIP3 is the easy target for degradation.

  To minimize protein degradation, cells (lanes 6-8) were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  Exposure time: Lanes 1-2, 6-8: 26 seconds; Lanes 3-5: 59 seconds

  All lanes: Western blot - Anti-RIP3 antibody [EPR26080-567] (Anti-RIP3 antibody [EPR26080-567] ab322555) at 1/1000 dilution

  Lanes 1 and 6: L-929 (mouse connective tissue fibroblast) whole cell lysate at 20 µg

  Lanes 2 and 7: Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

  Lane 3: J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage ) whole cell lysate at 20 µg

  Lane 4: RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

  Lane 5: B16-F10 (mouse skin melanoma cell) whole cell lysate at 20 µg

  Lane 8: J774A.1 (mouse reticulum cell sarcoma monocyte/macrophage ) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 53 kDa, 38 kDa, 15 kDa

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Low expression: spleen (PMID: 2171047).

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-DBI antibody [EPR28926-2] (Anti-DBI antibody [EPR28926-2] ab317823) at 1/1000 dilution

  Lane 1: Human liver tissue lysate at 20 µg with 5% NFDM/TBST

  Lane 2: Human cerebellum tissue lysate at 20 µg with 5% NFDM/TBST

  Lane 3: Human spleen tissue lysate at 20 µg with 5% NFDM/TBST

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Observed band size: 11 kDa, 36 kDa

  Exposure time: 180s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  The expression of phosphorylated FAK is upregulated in response to pervanadate treatment (PMID: 20847951).

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  In Western blot, Anti-FAK antibody [EP695Y] (Anti-FAK antibody [EP695Y] ab40794) staining at 1/1000 dilution.

  All lanes: Western blot - Anti-FAK (phospho Y397 + Y576 + Y925) antibody [RM1141] (Anti-FAK (phospho Y397 + Y576 + Y925) antibody [RM1141] ab322354) at 1/1000 dilution

  Lane 1: Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

  Lane 2: HeLa treated with 10μM pervanadate for 60 minutes whole cell lysate (untreated membrane) at 20 µg

  Lane 3: Untreated HeLa whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

  Lane 4: HeLa treated with 10μM pervanadate for 60 minutes whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 119 kDa, 15 kDa

  Exposure time: 8s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:31011167).

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  All lanes: Western blot - Anti-IRAK4 antibody [EPR26377-254] (Anti-IRAK4 antibody [EPR26377-254] ab318954) at 1/1000 dilution

  Lane 1: Rat colon tissue lysate at 20 µg

  Lane 2: Rat lymph node tissue lysate at 20 µg

  Lane 3: 2.4G2 (rat B cell lymphoma B lymphocyte) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 37 kDa, 52 kDa, 15 kDa

  Exposure time: 59s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (Anti-6X His tag® antibody [EPR20547] - ChIP Grade ab213204) staining at 1/5000 dilution.

  All lanes: Western blot - Anti-ROR1 antibody [6D4] (Anti-ROR1 antibody [6D4] ab321998) at 1/1000 dilution

  Lane 1: 293T (human embryonic kidney epithelial cell) transfected with an empty vector (vector control) containing a myc-His-tag® whole cell lysate at 20 µg

  Lane 2: 293T transfected with a ROR2 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

  Lane 3: 293T transfected with a ROR1 expression vector containing a myc-His-tag® whole cell lysate at 20 µg

  Secondary

  All lanes: Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

  Observed band size: 130 Da, 15 kDa, 46 kDa

  Exposure time: 1s

• 

  Western blot - Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  Low expression: liver (PMID: 25950489).

  The molecular weight observed is consistent with what has been described in the literature (PMID: 38514849). The ~38 kDa band likely represents a N-terminal cleavage product of RIP3.

  This antibody was tested on frozen and fresh mouse colon and mouse spleen tissue lysates and it was concluded that RIP3 is the easy target for degradation.

  To minimize protein degradation, tissues (lanes 4-6) were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

  In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) staining at 1/100000 dilution.

  Exposure time: Lanes 1-4 : 180 seconds; Lane 5 : 37 seconds; Lane 6: 70 seconds

  All lanes: Western blot - Anti-RIP3 antibody [EPR26080-567] (Anti-RIP3 antibody [EPR26080-567] ab322555) at 1/1000 dilution

  Lane 1: Mouse colon tissue lysate at 20 µg

  Lane 2: Mouse liver lysate at 20 µg

  Lanes 3 and 6: Mouse spleen tissue lysate at 20 µg

  Lane 4: Mouse testis tissue lysate at 40 µg

  Lane 5: Mouse colon tissue lysate at 40 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 53 kDa, 38 kDa, 15 kDa