Anti-SMCHD1 antibody [EPR27952-42]

• IP

Supplier Data

Immunoprecipitation - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

SMCHD1 was immunoprecipitated from 0.35 mg HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate with ab317811 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317811 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 2 : ab317811 IP in HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317811 in HCT 116 whole cell lysate

All lanes:

Immunoprecipitation - Anti-SMCHD1 antibody [EPR27952-42] (ab317811) at 1/30 dilution

All lanes:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• IP

Lab

Immunoprecipitation - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

SMCHD1 was immunoprecipitated from 0.35 mg EL4 (mouse lymphoma T lymphocyte) whole cell lysate with ab317811 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317811 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-SMCHD1 antibody [EPR27952-42] (ab317811) at 1/1000 dilution

Lane 1:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate (Input) at 10 µg

Lane 2:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab317811 in EL4 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 226 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane 1-4 : 180 seconds; Lane 5-8 : 10 seconds

In Western blot analysis, ab317811 exhibited lower sensitivity compared to ab179456, for which reason we recommend the latter as a superior alternative.

Protocol optimizations including used gels with concentration of 6% and a two hours electrical transfer are suggested if ab109746 must be used.

Lanes 1 - 4:

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (ab317811) at 1/1000 dilution

Lanes 5 - 8:

Western blot - Anti-SMCHD1 antibody [EPR12340-29] (<a href='/ko/products/primary-antibodies/smchd1-antibody-epr12340-29-ab179456'>ab179456</a>) at 1/1000 dilution

Lanes 1 and 5:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 2 and 6:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 3 and 7:

U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Lanes 4 and 8:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 226 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

Low expression : MCF7.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

This Western blot was performed using 6% gels, followed by a two-hour electrical transfer, which showed significantly stronger signals.

All lanes:

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (ab317811) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

SK-OV-3 (human ovarian cancer epithelial cell) whole cell lysate at 20 µg

Lane 8:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 226 kDa

false

Exposure time: 180s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HCT 116 (human colorectal carcinoma epithelial cell) cells and 5 µg of ab317811 [EPR27952-42]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HCT 116 (human colorectal carcinoma epithelial cell) cells and 5 µg of ab317811 [EPR27952-42]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10⁵ HCT 116 (human colorectal carcinoma epithelial cell) cells and 5 µg of ab317811 [EPR27952-42]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

Low expression : liver.

This Western blot was performed using 6% gels, followed by a two-hour electrical transfer, which showed significantly stronger signals.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (ab317811) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 50 µg

Lane 2:

Mouse testis tissue lysate at 50 µg

Lane 3:

Mouse liver tissue lysate at 50 µg

Secondary

All lanes:

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

Observed band size: 226 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

This Western blot was performed using 6% gels, followed by a two-hour electrical transfer, which showed significantly stronger signals.

All lanes:

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (ab317811) at 1/1000 dilution

Lane 1:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 50 µg

Lane 3:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 50 µg

Secondary

All lanes:

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

Observed band size: 226 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

Low expression : liver.

The lanes 4-5 of this blot was developed using a high sensitivity ECL substrate.

The identity of the lower MW bands in lanes 4-5 are unknown.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

Exposure time : Lanes 1-3 : 180 seconds; Lanes 4-5 : 103 seconds.

Lane 1:

Mouse spleen tissue lysate

Lane 2:

Mouse testis tissue lysate

Lane 3:

Mouse liver tissue lysate

Lane 4:

Rat spleen tissue lysate

Lane 5:

Rat liver tissue lysate

false

• WB

Supplier Data

Western blot - Anti-SMCHD1 antibody [EPR27952-42] (AB317811)

Low expression : MCF7.

The lane 9 of this blot was developed using a high sensitivity ECL substrate.

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

Exposure time : Lanes 1-8 : 180 seconds; Lane 9 : 59 seconds.

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Lane 2:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate

Lane 3:

U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate

Lane 6:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate 20

Lane 7:

SK-OV-3 (human ovarian cancer epithelial cell) whole cell lysate

Lane 8:

C2C12 (mouse myoblast) whole cell lysate

Lane 9:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate

false