Chromatin was prepared from Ramos cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab269454 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads.  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 commercial primers from paper: PMC2905439

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

 

Immunohistochemical analysis of paraffin-embedded Human diffuse large B-cell lymphoma tissue labeling AICDA with ab269454 at 1/4000 dilution (0.12ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mainly cytoplasmic staining (weak nuclear staining) in part of tumor cells of human diffuse large B-cell lymphoma (PMID: 29251015).

The section was incubated with ab255611 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NAMALWA (human Burkitt's lymphoma B lymphocyte) and K-562 (human chronic myelogenous leukemia lymphoblast) cells labelling AICDA with ab269454 at 1/50 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NAMALWA cell line. Negative control: K-562 cell line (PMID: 27217538). ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Ramos (human Burkitt's lymphoma B lymphocyte) cells labelling AICDA with ab269454 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human Hodgkin lymphoma tissue labeling AICDA with ab269454 at 1/4000 dilution (0.12ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mainly cytoplasmic staining (weak nuclear staining) in part of tumor cells of human Hodgkin lymphoma (PMID: 15732141).

The section was incubated with ab255611 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling AICDA with ab269454 at 1/4000 dilution (0.12ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mainly cytoplasmic staining (weak nuclear staining) in germinal center cells of human tonsil (PMID:23877718, 15732141, PMID: 29251015).

The section was incubated with ab255611 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

AICDA was immunoprecipitated from 0.35 mg NAMALWA (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab269454 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269454 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: NAMALWA whole cell lysate 10ug.

Lane 2: ab269454 IP in NAMALWA whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269454 in NAMALWA whole cell lysate.

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

Exposure time: 90 seconds.

 

AICDA was immunoprecipitated from 0.35 mg Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab269454 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269454 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Ramos whole cell lysate 10ug.

Lane 2: ab269454 IP in Ramos whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269454 in Ramos whole cell lysate.

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

Exposure time: 90 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized K-562 (human chronic myelogenous leukemia lymphoblast, Left) / NAMALWA (human Burkitt's lymphoma B lymphocyte, Right) cells labelling AICDA with ab269454 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: K-562 cell line (PMID: 27217538).

All lanes : Anti-AICDA antibody [EPR23436-45] - ChIP Grade (ab269454) at 1/1000 dilution

Lane 1 : Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate
Lane 2 : K-562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 3 : NAMALWA (human Burkitt's lymphoma B lymphocyte), whole cell lysate
Lane 4 : Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 23 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 114 seconds.

Negative control: K-562 (PMID: 27217538).

The expression profile observed is consistent with what has been described in the literature (PMID: 27217538).