Chromatin was prepared from ES-D3 (Mouse embryo spherical colony) 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 ab254336 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers are from paper PMID:20123894

 

Anti-MTF2 antibody [EPR23536-87] - ChIP Grade (ab254336) at 1/1000 dilution + Human testis tissue lysate at 20 µg

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

Predicted band size: 67 kDa
Observed band size: 47,56,67 kDa why is the actual band size different from the predicted?

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

Exposure time: 3 minutes.

 

MTF2 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab254336 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254336 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug

Lane 2: ab254336 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254336 in HeLa whole cell lysate

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

Exposure time: 24 seconds.

67, 56, 47, 29-kDa bands correspond to MTF2 isoforms.This blot was developed using a higher sensitivity ECL substrate.

All lanes : Anti-MTF2 antibody [EPR23536-87] - ChIP Grade (ab254336) at 1/1000 dilution

Lane 1 : Mouse skeletal muscle tissue lysate
Lane 2 : Mouse testis tissue lysate
Lane 3 : Rat testis tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 67 kDa
Observed band size: 47,56,67 kDa why is the actual band size different from the predicted?

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

Exposure time: 92 seconds.

 

MTF2 was immunoprecipitated from 0.35 mg ES-D3 (mouse embryonic multipotent stem Cell) whole cell lysate with ab254336 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254336 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: ES-D3 (mouse embryonic multipotent stem Cell) whole cell lysate 10 ug

Lane 2: ab254336 IP in ES-D3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254336 in ES-D3 whole cell lysate

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

Exposure time: 8 seconds.

67, 56, 47, 29-kDa bands correspond to MTF2 isoforms. Bands between 30-37 kDa is caused by degradation as demonstrated by WB data.

All lanes : Anti-MTF2 antibody [EPR23536-87] - ChIP Grade (ab254336) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lanes 3 & 6 : ES-D3 (mouse embryonic mtipotent stem Cell), whole cell lysate 20
Lane 4 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 5 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution

Predicted band size: 67 kDa
Observed band size: 47,56,67 kDa why is the actual band size different from the predicted?

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 28869966).

Lysate loaded onto lane 6 was made freshly and used in WB immediately to minimize protein degradation. 

67, 56, 47, 29-kDa bands correspond to MTF2 isoforms. Bands between 30-37 kDa in lane3 is caused by degradation.

 Exposure time: Lanes 1-2: 26 seconds; Lane 3: 10 seconds; Lanes 4-5: 125 seconds; Lane 6: 3 minutes.