Anti-c-Jun antibody [E254] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Jun antibody [E254] - ChIP Grade (AB32137)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling c-Jun with purified ab32137 at 1 : 100 dilution (1.2 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

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Immunoprecipitation - Anti-c-Jun antibody [E254] - ChIP Grade (AB32137)

ab32137 (purified) at 1/500 dilution (2.29 © : g/ml) immunoprecipitating c-Jun in HEK-293 whole cell lysate.
Lane 2 (+) : HEK-293(Human embronic kidney epithelial cell) whole cell lysate 10 © : g
Lane 3 (-) : ab32137 & HEK-293 whole cell lysateRabbit monoclonal IgG (ab172730) instead of ab32137 in HEK-293 whole cell lysate
For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1/1000 dilution.
Blocking and diluting buffer : 5% NFDM /TBST .

All lanes:

Immunoprecipitation - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

Predicted band size: 35 kDa

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• ChIP

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ChIP - Anti-c-Jun antibody [E254] - ChIP Grade (AB32137)

Chromatin was prepared from PC-12 (starve overnight) + NGF ( 50 ng/ml 2h) cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab32137 (blue), and 20µl of protein A/G sepharose beads slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 5µg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

• IP

Unknown

Immunoprecipitation - Anti-c-Jun antibody [E254] - ChIP Grade (AB32137)

c-Jun was immunoprecipitated using 0.5mg NIH3T3 whole cell extract, 5µg of Rabbit polyclonal to c-Jun 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, NIH3T3 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^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab32137.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : 45kDa; c-Jun

All lanes:

Immunoprecipitation - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137)

Predicted band size: 35 kDa

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• WB

Lab

Western blot - Anti-c-Jun antibody [E254] - ChIP Grade (AB32137)

Blocking and diluting buffer : 5% NFDM/TBST

For Lysate preparation protocol, please refer to the protocol section of the website and/or here.

All lanes:

Western blot - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137) at 1/200 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates prepared in RIPA lysis method at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates prepared in 1% SDS Hot lysis method. at 15 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates prepared in RIPA lysis method at 15 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates prepared in 1% SDS Hot lysis method 15ug. at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 39 kDa

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Exposure time: 3min

• WB

Lab

Western blot - Anti-c-Jun antibody [E254] - ChIP Grade (AB32137)

All lanes:

Western blot - Anti-c-Jun antibody [E254] - ChIP Grade (ab32137) at 1/5000 dilution

All lanes:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 43 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-c-Jun antibody [E254] - ChIP Grade (AB32137)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• IHC

CiteAb

Immunohistochemistry - Anti-c-Jun antibody [E254] - ChIP Grade (AB32137)

Immunohistochemistry-immunofluorescence using Anti-c-Jun antibody [E254] - ChIP Grade, ab32137. Publication image from Jacob, C. et al., 2017, Nat Commun, 28139683. Legend direct from paper.

Delayed Oct6 and earlier/higher cJun upregulation in dKO.Western blots and quantification normalized to GAPDH of (a) Oct6, (b) cJun, (c) Pax3, and (d) Sox2 in crushed (Cr) and contralateral (Co=1) nerves of control (Ctrl) and dKO, showing delayed Oct6 upregulation and earlier/higher cJun upregulation in dKO. Of note : the double band at ∼52 kDa and the lower band at ∼42 kDa detected by the Oct6 antibody were quantified and added together in the graph. Dashed lines : samples run on the same gel, but not on consecutive lanes. In a–d, coloured graphs quantify protein levels in Cr compared to Co, and grey/white bar graphs quantify protein levels in dKO compared with Ctrl crushed nerves. (e,g) Co-immunofluorescence of Oct6 (red, e) at 5 dpl or of cJun (green, g) at 1 dpl with the macrophage marker F4/80 (green in e, red in g) and DAPI labelling (nuclei) in control and dKO nerves showing decreased Oct6 and increased cJun levels in SCs of dKO compared with control nerves. White arrows indicate SCs (F4/80-negative cells with elongated nuclei) and blue arrowheads indicate macrophages (F4/80-positive cells). (f) Oct6 in situ hybridization in control and dKO nerves at 5 dpl showing decreased Oct6 transcript levels in dKO. Sample size : (a–d) 5 animals per group for 1 dpl, 2 dpl and 3 dpl, and 6 or 9 animals per group for 5 dpl and 12 dpl; (e–g) 3 animals per group. One-tailed (grey asterisk; blue/red asterisks : Control Oct6 at 5 dpl, Control cJun at 5 dpl, dKO cJun at 12 dpl, Control Pax3 at 2 dpl, Control Sox2 at 3 dpl and 5 dpl) or two-tailed (black asterisks; blue/red asterisks : other values) Student's t-tests, unpaired (bar graphs, and cJun at 2 dpl in coloured graph) or paired (coloured graphs, except for cJun at 2 dpl), P values : *P<0.05, **P<0.01. Values, mean; error bars, s.e.m. Scale bar, 10 µm.