Chromatin was prepared from MCF7 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 ab217678 (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.

The observed enrichment profile of Rad21 binding is consistent with what has been described in the literature (PMID: 23145106)

 

All lanes : Anti-Rad21 antibody [EPR22506-15] - ChIP Grade (ab217678) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 3 : C6 (rat glial tumor glial cell), whole cell lysate whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 72 kDa
Observed band size: 130-75 kDa why is the actual band size different from the predicted?

Full length Rad21 is approximately 130kDa, cleaved Rad21 is approximately 75 kDa. The molecular weight observed are consistent with what has been described in the literature (PMID: 12417729, PMID 21876002).

Exposure time: 7.7 seconds

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

All lanes : Anti-Rad21 antibody [EPR22506-15] - ChIP Grade (ab217678) at 1/1000 dilution

Lane 1 : Rat Brain tissue lysate
Lane 2 : Mouse Brain tissue lysate
Lane 3 : Mouse heart tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 72 kDa
Observed band size: 130-75 kDa why is the actual band size different from the predicted?

Full length Rad21 is approximately 130kDa, cleaved Rad21 is approximately 75 kDa. The molecular weight observed are consistent with what has been described in the literature (PMID: 12417729, PMID 21876002).

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 3 minutes.

 

All lanes : Anti-Rad21 antibody [EPR22506-15] - ChIP Grade (ab217678) at 1/1000 dilution

Lane 1 : Human heart tissue lysate
Lane 2 : Human fetal kidney tissue lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 3-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 72 kDa
Observed band size: 130-75 kDa why is the actual band size different from the predicted?

Full length Rad21 is approximately 130 kDa, cleaved Rad21 is approximately 75 kDa. The molecular weight observed are consistent with what has been described in the literature (PMID: 12417729, PMID 21876002).

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Exposure times:

Lanes1-2: 37 seconds

Lane 3: 5.5 seconds

Lane 4: 48 seconds

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Rad21 using ab217678 at 1/1000 dilution for 30 minutes at room temperature, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat colon (PMID: 25575569) is observed. Counterstained with Hematoxylin. 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 mouse stomach tissue labeling Rad21 using ab217678 at 1/1000 dilution for 30 minutes at room temperature, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse stomach (PMID: 25575569) is observed. Counterstained with Hematoxylin. 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 stomach tissue labeling Rad21 using ab217678 at 1/1000 dilution for 30 minutes at room temperature, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human stomach (PMID: 25575569) is observed. Counterstained with Hematoxylin. 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 Rad21 using ab217678 at 1/1000 dilution for 30 minutes at room temperature, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the human breast carcinoma (PMID: 21255398) is observed. Counterstained with Hematoxylin. 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.

Rad21 was immunoprecipitated from 0.35mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab217678 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab217678 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10µg (input)
Lane 2: ab217678 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab217678 in HeLa whole cell lysate.

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

Exposure time: 3 seconds.

Faint band below 75kDa in lane 2 could be Rad21 cleavage product as described in the literature (PMID: 28854249).

Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (mouse embryonic fibroblast) cell line labeling Rad21 using ab217678 at 1/500 dilution (red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling Rad21 using ab217678 at 1/500 dilution (red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling Rad21 with ab217678 at 1/100 dilution, followed by an AlexaFluor^®488 Goat anti-Rabbit secondary antibody (ab150077) at 1/1000 dilution (green). Confocal image showing nuclear staining in NIH/3T3 cell line is observed. The nuvlear counterstain is DAPI (Blue). Tubulin is counterstained using Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is an AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Rad21 with ab217678 at 1/100 dilution, followed by an AlexaFluor^®488 Goat anti-Rabbit secondary antibody (ab150077) at 1/1000 dilution (green). Confocal image showing nuclear staining in HeLa cell line is observed. The nuvlear counterstain is DAPI (Blue). Tubulin is counterstained using Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is an AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.