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

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/µL. 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 downloaded here.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

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).

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

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.

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.

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.

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.

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).

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. 

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.

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. 

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.