Anti-CTCF antibody [EPR18253] - ChIP Grade

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Immunohistochemical analysis of paraffin-embedded human endometrium tissue labeling CTCF with ab188408 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human endometrium is observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling CTCF with ab188408 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows :
-ve control 1 : ab188408 at 1/2000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : ab7291 Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• ChIP-seq

Lab

ChIP-sequencing - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 μg of chromatin and 4 μg of ab188408. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. ChIP-Seq validation performed with ChIP-Kit Transcription Factors ChIP-Seq (ab270813). Additional screenshots of mapped reads can be downloaded here.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 30 μg of chromatin and 4 μg of Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408). ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed by Active Motif, Carlsbad, CA. Additional screenshots of mapped reads can be downloaded here.

• ChIP

Unknown

ChIP - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Chromatin was prepared from HeLa (Human epithelial cells from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 2μg of ab188408 (blue), and 20μl of Anti Rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyronic fibroblast cells) cells labeling CTCF with ab188408 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) seconday antibody at 1/1000 dilution (red).
The negative controls are as follows :
-ve control 1 : ab188408 at 1/2000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Immunohistochemical analysis of paraffin-embedded rat stomach tissue labeling CTCF with ab188408 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on the epithelium cells of rat stomach is observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling CTCF with ab188408 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on hepatocytes of mouse liver is observed. Counter stained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Chromatin was prepared from Mouse Embryonic Fibroblast cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 8 µg of [Anti-CTCF antibody]( ab188408). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section.

• WB

Supplier Data

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Blocking/Dilution buffer : 5% NFDM/TBST.

Observed MW : 140, 130, 97, 80, 73, 70, 55 kDa.

The multiples bands observed in the WB have been reported in literature (PMID : 12878173, PMID : 8246978).

All lanes:

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 83 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Blocking/Dilution buffer : 5% NFDM/TBST.

Observed MW : 140, 97, 80, 73, 70, 55 kDa.

The multiples bands observed in the WB have been reported in literature (PMID : 12878173, PMID : 8246978).

All lanes:

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408) at 1/5000 dilution

Lane 1:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

LLC (Mouse lung carcinoma) whole cell lysate at 20 µg

Lane 3:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

Lane 4:

HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysate at 20 µg

Lane 5:

HepG2 (Human liver hepatocellular carcinoma) whole cell lysate at 20 µg

Lane 6:

MCF7 (Human breast adenocarcinoma cell line) 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 1/100000 dilution

Predicted band size: 83 kDa

false

Exposure time: 1s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 105 HeLa cells and 5μg of ab188408 [EPR18253]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 4 μg of ab188408 [EPR18253]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be downloaded here.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5 µg of ab188408 [EPR18253]. 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 ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 HeLa cells and 8 µg of ab188408. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Supplier Data

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Blocking/Dilution buffer : 5% NFDM/TBST.

Observed MW : 140, 130, 97, 80, 73, 70, 55 kDa.

The multiples bands observed in the WB have been reported in literature (PMID : 12878173, PMID : 8246978).

All lanes:

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Mouse kidney lysate at 10 µg

Lane 4:

Mouse spleen lysate at 10 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 1/100000 dilution

Predicted band size: 83 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Blocking/Dilution buffer : 5% NFDM/TBST.

Observed MW : 140, 130, 97, 80, 73, 70, 55 kDa.

The multiples bands observed in the WB have been reported in literature (PMID : 12878173, PMID : 8246978).

All lanes:

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor cells) whole cell lysate at 10 µg

Lane 2:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 3:

NIH/3T3 (Mouse embryonic fibroblast cells) whole cell lysate at 10 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 1/100000 dilution

Predicted band size: 83 kDa

false

Exposure time: 1s

• WB

Unknown

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

Blocking/Dilution buffer : 5% NFDM/TBST.

Observed MW 140, 130, 97, 80, 73, 70, 55 kDa.

The multiples bands observed in the WB have been reported in literature (PMID : 12878173, PMID : 8246978).

All lanes:

Western blot - Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408) at 1/1000 dilution

Lane 1:

Rat brain lysate at 10 µg

Lane 2:

Rat heart lysate at 10 µg

Lane 3:

Rat kidney lysate at 10 µg

Lane 4:

Rat spleen lysate at 10 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 1/100000 dilution

Predicted band size: 83 kDa

false

Exposure time: 2s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 F9 (Mouse embryonic testicular cancer cell line) cells and 5µg of ab188408 [EPR18253]. 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. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• CUT&Tag-seq

Supplier Data

CUT&Tag sequencing - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

CUT&Tag-seq was performed using 200,000 Oli-neu (Oligodendrocyte progenitor) cells. Cells were permeabilized with 0.05% Digitonin and 0.01% NP-40 for 3 minutes. A 1 : 100 dilution of Recombinant Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408) was used, along with a Guinea pig anti-rabbit Secondary. DNA was seg using Illumina NovaSeq S Prime to a depth of 24 million reads.

This image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.

This experiment and image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.

• CUT&Tag

Supplier Data

CUT&Tag - Anti-CTCF antibody [EPR18253] - ChIP Grade (AB188408)

CUT&Tag-seq was performed using 200,000 Oli-neu (Oligodendrocyte progenitor) cells. Cells were permeabilized with 0.05% Digitonin and 0.01% NP-40 for 3 minutes. A 1 : 100 dilution of Recombinant Anti-CTCF antibody [EPR18253] - ChIP Grade (ab188408) was used, along with a Guinea pig anti-rabbit Secondary. DNA was seg using Illumina NovaSeq S Prime to a depth of 24 million reads. This image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

This experiment and image is courtesy of Dr Marek Bartosovic, Gonçalo Castelo-Branco Group, Karolinska Institutet.