All lanes : Anti-c-Myc antibody [Y69] (ab32072) at 1/1000 dilution

Lane 1 : Wild-type Jurkat cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : Wild-type HEK-293T cell lysate
Lane 4 : MYC knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 49 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab32072 observed at 57 kDa. Red - loading control, ab8245 observed at 37 kDa.

 ab32072 was shown to react with MYC in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab256500 (knockout cell lysate ab263850) was used. Wild-type and MYC knockout samples were subjected to SDS-PAGE. ab32072 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

c-Myc was immunoprecipitated using 0.5mg Jurkat (human T cell leukemia cell line from peripheral blood) whole cell extract, 5µg of unpurified rabbit monoclonal to c-Myc [Y69] and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with unpurified ab32072.

Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

Band: 57kDa; c-Myc [Y69]

ab32072 staining MYC in wild-type HEK293 cells (top panel) and MYC knockout HEK293 cells (ab256500) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32072 at 5μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

Photomicrographs of select tumors and reactive tissue stained for c-Myc (positive staining  is the brown nuclei). Positive control (Burkitt lymphoma with a confirmed c-Myc translocation) revealed uniform, intense staining in >90% of tumor cells (Burkitt). In contrast, reactive lymphoid tissue revealed variable staining in only 10% of normal lymphocyte nuclei (Tonsil). Representative images from Diffuse large B cell lymphoma (DLBCL) cases and associated percent c-Myc+ tumor nuclei: Case 1, 90% MYC+; Case 7, 70% MYC+; and Cases 35 and 38, 30% c-Myc+. c-Myc staining was exclusively nuclear in all cases under the described staining conditions.

All lanes : Anti-c-Myc antibody [Y69] (ab32072) at 1/1000 dilution

Lane 1 : Rat pancreas lysates
Lane 2 : AR42J (Rat pancreatic tumor epithelial cell) whole cell lysates
Lane 3 : Rat-1 (Rat embryonic fibroblast) whole cell lysates
Lane 4 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 5 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 20000 µg/ml

Predicted band size: 49 kDa

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

Lanes 1 and 2:  80 seconds exposure time 

Lanes 3 to 5: 5 seconds exposure time 

Observed MW: 57 KDa

Western blot of L363 MM cell and CA46 cells measuring expresison of c-Myc (using ab32072) and GAPDH as the dose of DC-34 increases. 

c-Myc protein levels are inhibited as a function of the dose of DC-34 in L363 cells; only the highest dose of DC-34 affected c-Myc in the more resistant CA46 Burkitt’s lymphoma cells.

ab32072 staining c-Myc in HeLa (human epithelial cell line from cervix adenocarcinoma) cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab32072 at 10μg/ml dilution (shown in green) and ab195889, mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Human colorectal carcinoma (CRC) tissues stained for c-Myc using ab32072 at 1/100 dilution in immunohistochemical analysis.

Panel A: c-Myc positive IHC staining.

Panel B: c-Myc negative IHC staining.

For the full image see PMID 24503701.

IHC image of ab32072 staining c-Myc in human adenocarcinoma formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32072, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

IHC image of ab32072 staining c-Myc in human esophagus formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab32072, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the Secondary only control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Expression of c-Myc, as determined by immunohistochemical staining of glioblastoma sample (left) and low-grade glioma tumor (right) with ab32072. Representative samples are shown. Scale bars  = 20 µm. Nuclei were counterstained with hematoxylin (in blue).

For the full image see PMID 25050814.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human diffuse large B cell lymphoma tissue labelling c-Myc with purified ab32072 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human adenocarcinoma of the colon tissue labelling c-Myc with purified ab32072 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human adenocarinoma of colon tissue labelling c-Myc with unpurified ab32072.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung adenocarinoma tissue labelling c-Myc with unpurified ab32072.

All lanes : Anti-c-Myc antibody [Y69] (ab32072) at 1/1000 dilution (unpurified)

Lane 1 : Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate
Lane 2 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate
Lane 3 : THP1 (Human acute monocytic leukemia cell line) Whole Cell Lysate
Lane 4 : A20 (Mouse B lymphoma cell line) Whole Cell Lysate
Lane 5 : RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 49 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?

The predicted molecular weight of c-Myc is 48 kDa (SwissProt), however we expect to observe a banding pattern at 57 kDa.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 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 ab32072 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP antibody, and visualised using ECL development solution ab133406.

All lanes : Anti-c-Myc antibody [Y69] (ab32072) at 1/1000 dilution

Lane 1 : MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysates
Lane 3 : K562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates
Lane 4 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates
Lane 5 : THP-1 (Human monocytic leukemia monocyte) whole cell lysates
Lane 6 : Rat spleen whole cell lysates
Lane 7 : L6 (Rat skeletal muscle myoblast) whole cell lysates
Lane 8 : Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysates
Lane 9 : RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 49 kDa
Observed band size: 57 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

Immunocytochemistry/immunofluorescence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling c-Myc with purified ab32072 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/100) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) (1/500).

Unpurified ab32072 staining c-Myc in HEK293 cells transfected with CACNB4-c-Myc by immunocytochemistry/ immunofluorescence.Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100 then blocked using 5% serum for 20 minutes at 25°C. Samples were then incubated with ab32072 at a 1/250 dilution for 16 hours at 4°C. The secondary used was an Alexa Fluor^® 488 conjugated goat anti-rabbit polyclonal, used at a 1/500 dilution.

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ICC/IF image of unpurified ab32072 stained HeLa (human epithelial cell line from cervix adenocarcinoma) cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32072, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

 

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with ab32072 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32072, 1/76 dilution) for 30 min at 22ºC. The secondary antibody used was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG [EPR25A] (monoclonal) (ab172730, 1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 nm bandpass filter. 

Equilibrium disassociation constant (K_D)
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