Immunocytochemistry/ Immunofluorescence - Rad21 antibody (ab42478)

ab42478 (1/200) staining Rad21 in assynchronous HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100 and counterstained with DAPI in order to highlight the nucleus (red). for further experimental details please see Abreview.

Image courtesy of an Abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

Western blot - Rad21 antibody (ab42478)

All lanes : Anti-Rad21 antibody (ab42478) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 Human embryonic kidney cell line Whole Cell Lysate
Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 75 kDa
Observed band size : 100 kDa (why is the actual band size different from the predicted?)
Additional bands at : 55 kDa (possible cleavage fragment).

Although the predicted band size is 75kDa based on Swiss-prot data, a band of 120kDa has been previously observed. Mol Cell Biol. 2002 Dec;22(23):8267-77 PMID: 12417729. The band seen at 100kDa is also consistent with the banding pattern observed for other commercially available antibodies to Rad21.

Western blot - Rad21 antibody (ab42478)

All lanes : Anti-Rad21 antibody (ab42478) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : Testis (Mouse) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 75 kDa
Observed band size : 120 kDa (why is the actual band size different from the predicted?)
Additional bands at : 55 kDa,85 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 4 minutes

Although the predicted band size is 75kDa based on Swiss-prot data, a band of 120kDa has been previously observed. Mol Cell Biol. 2002 Dec;22(23):8267-77 PMID: 12417729.

Immunoprecipitation - Anti-Rad21 antibody (ab42478)

Rad21 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Rad21 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab42478.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: 100kDa: Rad21; Non specific - 50kDa: We are unsure as to the identity of this extra band. Although the predicted band size is 75kDa based on Swiss-prot data, a band of 120kDa has been previously observed. Mol Cell Biol. 2002 Dec;22(23):8267-77 PMID: 12417729. The band seen at 100kDa is also consistent with the banding pattern observed for other commercially available antibodies to Rad21.

Immunocytochemistry/ Immunofluorescence - Rad21 antibody (ab42478)

ICC/IF image of ab42478 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab42478, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).