Anti-EED antibody [EPR23043-5] - BSA and Azide free

• ChIP

Unknown

ChIP - Anti-EED antibody [EPR23043-5] - BSA and Azide free (AB264566)

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)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, glycerol, azide and BSA (ab240650).

• IP

Supplier Data

Immunoprecipitation - Anti-EED antibody [EPR23043-5] - BSA and Azide free (AB264566)

This data was developed using ab240650, the same antibody clone in a different buffer formulation.EED was immunoprecipitated from 0.35 mg K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate 10ug 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 10ug

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 minutes

All lanes:

Immunoprecipitation - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566)

Predicted band size: 50 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-EED antibody [EPR23043-5] - BSA and Azide free (AB264566)

EED was immunoprecipitated from 0.35 mg K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab240650 at 1/30 dilution (2μg 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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, glycerol, azide and BSA (ab240650).

All lanes:

Immunoprecipitation - Anti-EED antibody [EPR23043-5] - ChIP Grade (<a href='/en-us/products/primary-antibodies/eed-antibody-epr23043-5-chip-grade-ab240650'>ab240650</a>)

Predicted band size: 50 kDa

false

• WB

Lab

Western blot - Anti-EED antibody [EPR23043-5] - BSA and Azide free (AB264566)

This data was developed using ab240650, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 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 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.

Lanes 1-3 : 15 seconds; Lanes 4-6 : 37 seconds.

Exposure time :

All lanes:

Western blot - Anti-EED antibody [EPR23043-5] - BSA and Azide free (ab264566) at 1/1000 dilution

Lanes 1 - 2:

293T (human embryonic kidney epithelial cell) 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

Secondary

All lanes:

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

Predicted band size: 50 kDa

false

• WB

Lab

Western blot - Anti-EED antibody [EPR23043-5] - BSA and Azide free (AB264566)

This data was developed using ab240650, the same antibody clone in a different buffer formulation.

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

ab240650 detects an unknown band close to the target bands in cytoplasm.

All lanes:

Western blot - Anti-EED antibody [EPR23043-5] - ChIP Grade (<a href='/en-us/products/primary-antibodies/eed-antibody-epr23043-5-chip-grade-ab240650'>ab240650</a>) at 1/1000 dilution

Lane 1:

Wild type HAP1 whole cell lysate at 20 µg

Lane 2:

EED knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Wild type HAP1 nuclear fraction lysate at 20 µg

Lane 4:

EED knockout HAP1 nuclear fraction lysate at 20 µg

Lane 5:

Wild type HAP1 cytoplasmic lysate at 20 µg

Lane 6:

EED knockout HAP1 cytoplasmic 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: 50 kDa

Observed band size: 50-70 kDa

false

Exposure time: 3s

• WB

Lab

Western blot - Anti-EED antibody [EPR23043-5] - BSA and Azide free (AB264566)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, glycerol, azide and BSA (ab240650).

All lanes:

Western blot - Anti-EED antibody [EPR23043-5] - ChIP Grade (<a href='/en-us/products/primary-antibodies/eed-antibody-epr23043-5-chip-grade-ab240650'>ab240650</a>) at 1/1000 dilution

Lane 1:

Wild type HAP1 whole cell lysate at 20 µg

Lane 2:

EED knockout HAP1 whole cell lysate at 20 µg

Lane 3:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg

Lane 4:

293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 5:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Lane 6:

C2C12 (mouse myoblasts myoblast), 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: 50 kDa

Observed band size: 50-70 kDa

false