Anti-SP1 antibody [EPR22648-50] - ChIP Grade

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Immunohistochemical analysis of paraffin-embedded human gastric carcinoma tissue labeling SP1 using ab231778 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counterstained with hematoxylin.

Nuclear staining on tumor cells of human gastric carcinoma (PMID : 14695137). The section was incubated with ab231778 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling SP1 using ab231778 at 1/500 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Counterstained with hematoxylin.

Nuclear staining on tumor cells of human breast carcinoma (PMID : 14695137). The section was incubated with ab231778 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling SP1 (green) with ab231778 at 1/100 dilution, followed by an AlexaFluor^® 488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Confocal image showing nuclear staining in HeLa cell line. The nuclear counterstain is DAPI (Blue). Tubulin was stained using an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (Red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a AlexaFluor^® 488 Goat anti-Rabbit secondary (ab150077).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SP1 using ab231778 at 1/60 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and a unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). The secondary antibody was a Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at a 1/2000 dilution.

• ChIP-seq

Lab

ChIP-sequencing - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

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 ab231778. 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-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Chromatin was prepared from Human SP1 knockout HeLa cell line (ab265519) cells overexpressing either mNeonGreen or mNeonGreen-SP1. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 μg ab321887 [EPR28835-76]. Assay quality control was conducted using 8 μg anti-SP1 (ab231778) on the same cell lines. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Chromatin was prepared from Human SP1 knockout HeLa cell line (ab265519) cells overexpressing either mNeonGreen or mNeonGreen-SP1. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 μg ab321887 [EPR28835-76]. Assay quality control was conducted using 8 μg anti-SP1 (ab231778) on the same cell lines. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP

Unknown

ChIP - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min.

The ChIP was performed with 25 μg of chromatin, 5 μg of ab231778 (red), and 20 μl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (sybr green approach).

Primers and probes are located in the first kb of the transcribed region.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

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-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778). 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.

• IP

Unknown

Immunoprecipitation - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

SP1 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate using ab231778 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab231778 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (input).
Lane 2 : ab231778 IP in HeLa whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab231778 in HeLa whole cell lysate.

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778)

Predicted band size: 81 kDa

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• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Chromatin was prepared from Human SP1 knockout HeLa cell line (ab265519) cells overexpressing either mNeonGreen or mNeonGreen-SP1. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ cells and 8 μg ab321887 [EPR28835-76]. Assay quality control was conducted using 8 μg anti-SP1 (ab231778) on the same cell lines. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• WB

Lab

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Lanes 1 and 3 : Samples were made from freeze-thaw cycled lysate.

Lanes 2 and 4 : Samples were freshly made and used immediately to minimize protein degradation.

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778) at 1/10000 dilution

Lanes 1 - 2:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Lanes 3 - 4:

293T (human embryonic kidney epithelial cell) whole cell lysate at 15 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: 81 kDa

Observed band size: 98 kDa

false

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

ab231778 was shown to specifically react with SP1 in wild-type HAP1 cells as signal was lost in SP1 knockout cells. Wild-type and SP1 knockout samples were subjected to SDS-PAGE. ab231778 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 blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

Degraded fragments (29-97kDa) of SP1 have been descried in the literature (PMID : 10329728).

All lanes:

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SP1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

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

Predicted band size: 81 kDa

Observed band size: 98 kDa

false

Exposure time: 8s

• WB

Lab

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Lanes 1-2 : Merged signal (red and green). Green - ab231778 observed at 100 kDa. Red - loading control ab8245 observed at 37 kDa.

ab231778 Anti-SP1 antibody [EPR22648-50] was shown to specifically react with SP1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265519 (knockout cell lysate ab257698) was used. Wild-type and SP1 knockout samples were subjected to SDS-PAGE. ab231778 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

SP1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human SP1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-sp1-knockout-hela-cell-line-ab265519'>ab265519</a>)

Predicted band size: 81 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

Western blot : Rabbit Monoclonal[EPR22648-50] to SP1 ab231778 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 90 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in SP1 knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human SP1 knockout MCF7 cell line (ab287770) at 20 µg

Lane 3:

HeLa at 20 µg

Lane 4:

Ramos at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 81 kDa

Observed band size: 90 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

ChIC/CUT&RUN was performed using pAG-MNase at a final concentration of 700 ng/mL. 2.5 x 10⁵ Human SP1 knockout HeLa cells (ab265519) over expressing either mNeonGreen or mNeonGreen-SP1 were used along with 5 μg of ab321887[EPR28835-76]. Assay quality control was conducted using 5 μg anti-SP1(ab231778) on the same cell lines. 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-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL. 2.5X10^5 of Human wild-type HeLa cell line (ab255928) or Human SP1 knockout HeLa cell line (ab265519) were used along with 5µg of ab231778 [EPR22648-50]. Assay Quality Control was conducted using 5µg Anti-CTCF (ab188408) on the same cell lines. 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.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

ChIC/CUT&RUN was performed using pAG-MNase at a final concentration of 700 ng/mL. 2.5 x 10⁵ Human SP1 knockout HeLa cells (ab265519) over expressing either mNeonGreen or mNeonGreen-SP1 were used along with 5 μg of ab321887[EPR28835-76]. Assay quality control was conducted using 5 μg anti-SP1(ab231778) on the same cell lines. 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-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

ChIC/CUT&RUN was performed using pAG-MNase at a final concentration of 700 ng/mL. 2.5 x 10⁵ Human SP1 knockout HeLa cells (ab265519) over expressing either mNeonGreen or mNeonGreen-SP1 were used along with 5 μg of ab321887[EPR28835-76]. Assay quality control was conducted using 5 μg anti-SP1(ab231778) on the same cell lines. 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

Supplier Data

Western blot - Anti-SP1 antibody [EPR22648-50] - ChIP Grade (AB231778)

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

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

In Western blot, Anti-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-mNeonGreen antibody [EPR28835-76] (<a href='/en-us/products/primary-antibodies/mneongreen-antibody-epr28835-76-ab321887'>ab321887</a>) at 1/1000 dilution

Lane 1:

SP1 knockout HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SP1 knockout HeLa cells transfected with a mNeonGreen-GPGPG-SP1 expression vector containing no His-tag&reg;, whole cell lysate at 20 µg

Lane 3:

SP1 knockout HeLa cells transfected with a mNeonGreen expression vector containing a myc-His-tag&reg;, 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

Observed band size: 36 kDa,133 kDa,35 kDa

false

Exposure time: 81s