All lanes : Anti-PAX5 antibody [SP34] - ChIP Grade (ab227635) at 0.172 µg/ml

Lane 1 : Ramos (Human Burkitt's lymphoma B lymphocyte) cell lysate
Lane 2 : Daudi (Human Burkitt's lymphoma lymphoblast) cell lysate
Lane 3 : Jurkat (Human T cell leukemia T lymphocyte) cell lysate
Lane 4 : U-937 (Human histiocytic lymphoma monocyte) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 0.05 µg
Lanes 2-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 0.05 µg/ml

Predicted band size: 42 kDa



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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat spleen tissue sections labeling PAX5 with ab227635 at 1/50 dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 6.0, epitope retrieval solution 1) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Nuclear staining on the rat spleen, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab228136 for 30 mins at room temperature.

Flow Cytometry analysis of Ramos(Human Burkitt's lymphoma B lymphocyte) cells labeling PAX5 with purified ab227635 at 1:20 dilution (8.6 µg/ml) Red. Cells were fixed with 4% paraformaldehyde . A Goat anti rabbit IgG (Alexa Fluor® 488, ab150081) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (ab172730) / Black. Unlabeled control - Unlabelled cells / Blue.

Chromatin was prepared from Ramos cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab227635 (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 (PMID: 19806612)
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

ab227635 (purified) at 1/20 dilution immunoprecipitating PAX5 in Ramos whole cell lysate.

Lane 1 (input): Ramos(Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab227635 & Ramos whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab227635 in Ramos whole cell lysate

For western blotting, ab227648 (purified at 1/500 dilution (0.344 µg/ml) and veriBlot for IP secondary antibody (HRP) (ab131366) at 1/1000 dilution were used.

Blocking and diluting buffer: 5% NFDM /TBST .

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling PAX5 with ab227635 at 1/50 dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 6.0, epitope retrieval solution 1) for 10mins. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Hematoxylin was used as a counterstain. Nuclear staining on the human tonsil, performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab228136 for 30 mins at room temperature.

Immunohistochemical analysis of Human tonsil tissue labeling PAX5 using ab227635.

Immunohistochemical analysis of Human B cell lymphoma tissue staining PAX5 using ab227635.