Chromatin was prepared from HUVEC cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with: 25 µg of chromatin, 5 µg of ab211493 (red), and 20 µl of protein A/G sepharose beads slurry (10 µl of sepharose A beads + 10 µl of sepharose G beads). 5 μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (SYBR green chemistry).

ChIP was performed according to the literature (PMID: 26923194).

All lanes : Anti-MEF2C antibody [EPR19089-202] - ChIP Grade (ab211493) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 3 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 4 : Mouse brain lysate
Lane 5 : Rat brain lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-3 & 5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Lane 4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 51 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?

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

Exposure time:

Lanes 1-2,4: 5 minutes

Lane 3: 15 seconds

Lane 5: 26 seconds

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilsed Jurkat (Human T cell leukemia T lymphocyte, Left) / Raji (Human Burkitt's lymphoma B lymphocyte, Right) cell lines labelling MEF2C with ab211493 at 1/500 dilution (Red) compared with the isotype control Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG Alexa Fluor® 488 (ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative control: Jurkat (PMID: 27876533).

All lanes : Anti-MEF2C antibody [EPR19089-202] - ChIP Grade (ab211493) at 1/1000 dilution

Lane 1 : Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate 20 µg
Lane 2 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate 20 µg
Lane 3 : Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate 20µg
Lane 4 : Daudi (human Burkitt's lymphoma lymphoblast), whole cell lysate 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 51 kDa
Observed band size: 50-60 kDa why is the actual band size different from the predicted?

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

Exposure time: 92 seconds.

The expression profile observed is consistent with the literature (PMID: 18450586). Negative control: Jurkat (PMID: 27876533)

All lanes : Anti-MEF2C antibody [EPR19089-202] - ChIP Grade (ab211493) at 1/1000 dilution

Lane 1 : Human MEF2C recombinant protein (aa271-493) 10 ng
Lane 2 : Human MEF2A recombinant protein (aa1-499, ab204772) 10 ng

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

Predicted band size: 51 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST
Exposure time: 3 seconds. 

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labelling MEF2C with ab211493 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in B lymphocytes of rat spleen but not in T cells in the periarterial lymphatic sheath is observed (PMID: 8506376, PMID 15703219). Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP)ready to use.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labelling MEF2C with ab211493 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in B lymphocytes of mouse spleen but not in T cells in the periarterial lymphatic sheath is observed (PMID: 8506376, PMID 15703219). Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP)ready to use.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue labelling MEF2C with ab211493 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in leukocytes but not in tumor cells of human endometrial carcinoma is observed (PMID: 8506376, PMID 15703219). Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP)ready to use.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labelling MEF2C with ab211493 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining in human skeletal muscle cells is observed(PMID: 8506376, PMID 15703219). Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP)ready to use.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilised Raji (human Burkitt's lymphoma B lymphocyte) cells labelling MEF2C with ab211493 at 1/500 dilution, followed by AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution (green). Confocal image showing nuclear staining in Raji cell line. Negative control: Jurkat (PMID: 27876533). DAPI was used as the nuclear counterstain, and the Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889 antibody was used as a counterstain at 1/200 dilution.

The negetive controls are as follows:
-ve control 1: ab197070 on jurkat (human T cell leukemia cell line from peripheral blood) cells.
-ve control 2: Jurkat cells stained with DAPI.
-ve control 3: Merged negetive contol images.