All lanes : Anti-EED antibody [EPR23043-5] - ChIP Grade (ab240650) at 1/1000 dilution

Lane 1 : Wild type HAP1 whole cell lysate
Lane 2 : EED knockout HAP1 whole cell lysate
Lane 3 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 4 : 293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 5 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 6 : C2C12 (mouse myoblasts myoblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 50 kDa
Observed band size: 50-70 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBST ab240650 was shown to specifically react with EED in wild-type HAP1 cells as signal was lost in EED knockout cells. Wild-type and EED knockout samples were subjected to SDS-PAGE. ab240650 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:27578866, 9584199). EED cDNA encodes 441-aa-long protein and 535-aa-long protein. 

Exposure time: Lanes 1-3: 15 seconds Lanes 4-6: 37 seconds

EED was immunoprecipitated from 0.35 mg K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab240650 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240650 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 µg

Lane 2: ab240650 IP in K562 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab240650 in K562 whole cell lysate

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

Exposure time: 1 min

 

Chromatin was prepared from NT2/D1 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 ab240650 (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 Millipore (Cat. No.: 17-10034) and CST (85322S)

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol

ChIP sequencing analysis of chromatin from Mouse Embryonic Stem Cells with ab240650 at 0.1 µL/µg chromatin. Cross linking was performed for 10 minutes with 1% PFA. Primary incubation was for 16 hours at 4°C in a dilution buffer containing 20mM Tris at pH8, 1.1mM EDTA, 1.1% triton, and 167mM NaCl.

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All lanes : Anti-EED antibody [EPR23043-5] - ChIP Grade (ab240650) at 1/1000 dilution

Lane 1 : Wild type Mouse Embryonic Stem Cell (mESC) cytoplasmic extract
Lane 2 : EED knockout (KO1) mESC cytoplasmic extract
Lane 3 : EED knockout (KO2) mESC cytoplasmic extract
Lane 4 : Wild type mESC nucleoplasm extract
Lane 5 : EED knockout (KO1) mESC nucleoplasm extract
Lane 6 : EED knockout (KO2) mESC nucleoplasm extract
Lane 7 : Wild type mESC chromatin extract
Lane 8 : EED knockout (KO1) mESC chromatin extract
Lane 9 : EED knockout (KO2) mESC chromatin extract

Secondary
All lanes : Goat anti-rabbit antibody conjugated to HRP at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 50 kDa
Observed band size: 40,47,55,60,65 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Additional bands at: 100 kDa, 70 kDa and 55 kDa in the cytoplasmic fraction (all possible non-specific binding)

This blot was produced using a 4-12% Bis-tris gel under reducing denaturing conditions. Following transfer, the membrane was blocked for 30 minutes at room temperature using 5% Milk before being incubated with ab240650 for 16 hours at 4°C.

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