Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free (AB240001)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SP1 with Purified ab124804 at 1 : 200 dilution (4 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with None. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124804)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free (AB240001)

ab124804, at 1/100 dilution staining SP1 in paraffin-embedded Human colon tissue, by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124804).

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

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free (AB240001)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SP1 with Purified ab124804 at 1/100 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124804)

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free (AB240001)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric carcinoma tissue sections labeling SP1 with Purified ab124804 at 1 : 2000 dilution (0.41 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Purified)ImmunoHistoProbe one step HRP Polymer (ready to use) was used for detection. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124804)

• WB

Lab

Western blot - Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free (AB240001)

This data was developed using ab124804, the same antibody clone in a different buffer formulation.

Western blot : Rabbit Monoclonal[EPR6662(B)] to SP1 ab124804 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 [EPR6662(B)] (<a href='/en-us/products/primary-antibodies/sp1-antibody-epr6662b-ab124804'>ab124804</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

SP1 knockout 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

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• WB

Lab

Western blot - Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free (AB240001)

This data was developed using the same antibody clone in a different buffer formulation (ab124804).

Lanes 1-2 : Merged signal (red and green). Green - ab124804 observed at 100 kDa. Red - loading control ab8245 observed at 37 kDa.

ab124804 Anti-SP1 antibody [EPR6662(B)] 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. ab124804 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 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 [EPR6662(B)] (<a href='/en-us/products/primary-antibodies/sp1-antibody-epr6662b-ab124804'>ab124804</a>) at 1/5000 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

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free (AB240001)

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 ab124804 [EPR6662(B)]. 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. This data was developed using the same antibody clone in a different buffer formulation (ab124804).

• OI-RD Scanning

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OI-RD Scanning - Anti-SP1 antibody [EPR6662(B)] - BSA and Azide free (AB240001)

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.