Anti-CTCF antibody [EPR7314(B)] - ChIP Grade

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Overlay histogram showing Hela cells stained with un-purified ab128873 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab128873, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Immunocytochemistry/immunofluorescence staining of 4% paraformaldehyde fixed; 0.1% triton X 100 permeabilized HeLa (human cervix adenocarcinoma) cells with purified ab128873 at dilution of 1/500. The secondary antibody used was Alexa Fluor® 488; goat anti-rabbit IgG (ab150077) at a dilution of 1/1000. Nucleus was counter-stained with DAPI (blue). ab7291, a mouse anti-tubulin antibody (1/1000) was used to stain tubulin along with ab150120 (AlexaFluor®594 goat anti-mouse secondary, 1/1000) shown in the top right hand panel. The negative controls are shown in the bottom middle and right hand panels- for negative control 1 rabbit primary antibody and anti-mouse secondary antibody (ab150120) was used. For negative control 2 mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) was used.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Immunohistochemical staining of paraffin-embedded human breast carcinoma sections labelling CTCF with purified ab128873 at dilution of 1 : 1000. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Flow cytometry analysis showing 4% paraformaldehyde fixed 293T (human embryonic kidney) cells labelling CTCF with purified ab128873 at dilution of 1/40 followed by the secondary antibody; Alexa Fluor® 488 goat-anti-rabbit IgG at dilution of 1/500 (red line). A non-specific IgG antibody (rabbit monoclonal) was used as isotype control (black line). The blue line shows cells without incubation with primary antibody and secondary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Immunohistochemical analysis of paraffin embedded Human colon tissue labelling CTCF with un-purified ab128873 at 1/250 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling CTCF with un-purified ab128873 at 1/250 dilution.

• ChIP-seq

Lab

ChIP-sequencing - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

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 ab128873. ChIP DNA was sequenced on the Illumina NextSeq 500 to a depth of 30 million reads. ChIP-Seq validation performed with ChIP-Ki Transcription Factors ChIP-Seq (ab270813). Additional screenshots of mapped reads can be downloaded here.

• ChIP

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ChIP - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 5μg of ab128873 (red), and 20μl of protein A/G sepharose beads slurry (10μl of sepharose A beads + 10μl of sepharose G beads). 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Immunohistochemical staining of paraffin-embedded rat kidney sections labelling CTCF with purified ab128873 at dilution of 1 : 1000. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Immunohistochemical staining of paraffin-embedded mouse kidney sections labelling CTCF with purified ab128873 at dilution of 1 : 1000. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• WB

Supplier Data

Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

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

In Western blot, Anti-CTCF antibody [EPR7314 (B)] - ChIP Grade (ab128873) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-T7 tag® antibody [EPR28033-8] (<a href='/en-us/products/primary-antibodies/t7-tag-antibody-epr28033-8-ab317258'>ab317258</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) transfected with empty vector, whole cell lysate at 20 µg

Lane 2:

HeLa transfected with an Human CTCF expression vector containi a T7 tag®, 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

Observed band size: 150 kDa,36 kDa

false

Exposure time: 8s

• WB

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Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (ab128873) at 1/10000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney) 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: 29 kDa,83 kDa

Observed band size: 140 kDa,35 kDa

false

• WB

Unknown

Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

All lanes:

Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (ab128873) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 83 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 2 µg of ab128873 [EPR7314(B)]. 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 4 µg of ab128873. 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.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

ChIP was performed with 1x10^7 HeLa cells and 4 µg of ab128873. 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).

• WB

Unknown

Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (ab128873) at 1/10000 dilution

All lanes:

Rat heart 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: 148 kDa,23 kDa,35 kDa,52 kDa,60 kDa,83 kDa,95 kDa

Observed band size: 110 kDa,140 kDa,146 kDa,32 kDa,35 kDa,50 kDa,60 kDa

false

• WB

Unknown

Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (ab128873) at 1/10000 dilution

All lanes:

Mouse brain 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

Observed band size: 140 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• CUT&Tag-seq

Collaborator

CUT&Tag sequencing - Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (AB128873)

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 [EPR7314(B)] - ChIP Grade (ab128873) 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 [EPR7314(B)] - ChIP Grade (AB128873)

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 [EPR7314(B)] - ChIP Grade (ab128873) 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.