Chromatin was prepared from Daudi cells starved for 24h, then treated with hIL-4(100ng/ml, 15min) 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 ab235591 (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 Cell Signaling Technology (Cat. No.: #56554)
*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

All lanes : Anti-STAT6 (phospho Y641) antibody [EPR22599-52] - ChIP Grade (ab235591) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) serum starved for 24 hours, whole cell lysate at 10 µg
Lane 2 : RAW264.7 serum starved for 24 hours, then treated with 100 ng/ml mIL-4 for 15 minutes, whole cell lysate 10 ug

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 94 kDa
Observed band size: 110,94 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST Phosphorylation of STAT6 at Y641 can be induced by IL-4 treatment (PMID: 21411736,15044251) The molecular weight  observed is consistent with what has been described in the literature (PMID: 24708771)  Exposure time: 4 seconds

All lanes : Anti-STAT6 (phospho Y641) antibody [EPR22599-52] - ChIP Grade (ab235591) at 1/1000 dilution

Lane 1 : 2.4G2 (rat B cell lymphoma B lymphocyte) serum starved for 24 hours, whole cell lysate at 10 µg
Lane 2 : 2.4G2 serum starved for 24 hours, then treated with 100 ng/ml rIL-4 for 15 minutes, whole cell lysate 10 ug

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 94 kDa
Observed band size: 110,94 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST Phosphorylation of STAT6 at Y641 can be induced by IL-4 treatment (PMID: 21411736,15044251) The molecular weight  observed is consistent with what has been described in the literature (PMID: 24708771)  Exposure time: 15 seconds

All lanes : Anti-STAT6 (phospho Y641) antibody [EPR22599-52] - ChIP Grade (ab235591) at 1/1000 dilution

Lane 1 : Daudi (human Burkitts lymphoma lymphoblast) serum starved for 24 hours, whole cell lysate at 10 µg
Lane 2 : Daudi serum starved for 24 hours, then treated with 100 ng/ml IL-4 for 15 minutes, whole cell lysate 10 ug

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 94 kDa
Observed band size: 110,75,81 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST Phosphorylation of STAT6 at Y641 can be induced by IL-4 treatment (PMID: 21411736,15044251) This antibody recognizes three isoforms of STAT6, isoform 1 (110 kDa), isoform 2 (75 kDa), isoform 3 (81 k Da), which is consistent with the Uniprot annotation.  Exposure time: 4 seconds

Dot blot analysis using ab235591 at 1/1000 dilution, Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at 1/100,000 diluton.

Lane 1: STAT6 (phospho Y641) peptide (aa636-647).

Lane 2: STAT6 non-phospho peptide (aa636-647)

Exposure time: 26 seconds.

STAT6 (phospho Y641) was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate with ab235591 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab235591 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: RAW 264.7 cells serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate 10ug

Lane 2: ab235591 IP in RAW 264.7 cells serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab235591 in RAW 264.7 was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate

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

Exposure time: 10 seconds

 

STAT6 (phospho Y641) was immunoprecipitated from 0.35 mg Daudi (Human Burkitt's lymphoma lymphoblast) cells serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate, with ab235591 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab235591 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Daudi cells serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate 10ug

Lane 2: ab235591 IP in Daudi was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab235591 in Daudi cells serum starved for 24h, then treated with 100ng/ml IL-4 for 15min whole cell lysate

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

Exposure time: 10 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) cells was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min (Red) / Untreated control (Green) cells labelling STAT6 (phospho Y641) with ab235591 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.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Daudi (Human Burkitt's lymphoma lymphoblast) was serum starved for 24h, then treated with 100ng/ml IL-4 for 15min (Red) / Untreated control (Green) cells labelling STAT6 (phospho Y641) with ab235591 at 1/500 (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.