Western blot - BRIT1 antibody (ab2612)

Lanes 1 - 3 : Anti-BRIT1 antibody (ab2612) at 1/250 dilution
Lanes 4 - 6 : Anti-BRIT1 antibody (ab2612) at 1/500 dilution
Lanes 7 - 9 : Anti-BRIT1 antibody (ab2612) at 1/1000 dilution

Lane 1 : 30 ug of LCL1 whole cell lysate.
Lane 2 : 30 ug of LCL2 whole cell lysate.
Lane 3 : 30 ug of LCL3 whole cell lysate.
Lane 4 : 30 ug of LCL1 whole cell lysate.
Lane 5 : 30 ug of LCL2 whole cell lysate.
Lane 6 : 30 ug of LCL3 whole cell lysate.
Lane 7 : 30 ug of LCL1 whole cell lysate.
Lane 8 : 30 ug of LCL2 whole cell lysate.
Lane 9 : 30 ug of LCL3 whole cell lysate.


Performed under reducing conditions.

Predicted band size : 93 kDa
Observed band size : 95 kDa (why is the actual band size different from the predicted?)
Additional bands at : 180 kDa,60 kDa (possible cleavage fragment),70 kDa (possible cleavage fragment). We are unsure as to the identity of these extra bands.

Exposure time : 15 seconds

LCL1, LCL2, and LCL3 are three patient derived fibroblast cell lines. The middle band is BRIT1 (denoted by arrow) as determined by treatment with siRNA.

Western blot - BRIT1 antibody (ab2612)

All lanes : Anti-BRIT1 antibody (ab2612) at 1/1000 dilution

Lane 1 : GUS (beta glucuronidase) expression negative control.
Lane 2 : In vitro expressed human BRIT1

Secondary
A rabbit polyclonal to Goat IgG H&L (HRP) secondary antibody was used at a 1:5000 dilution.
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 93 kDa
Observed band size : 100 kDa (why is the actual band size different from the predicted?)


ab2612 was incubated for one hour at room temperature with the membrane in 5% non-fat dry milk plus PBST.

This image was submitted courtesy of I.Gavvovidis, D. Schindler, Institut fuer Humangenetik, Universitaet Wuerzburg

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - BRIT1 antibody (ab2612)

Image courtesy of Human Protein Atlas

ab2612 staining in human lung, showing staining of the surface epithelial cells (in brown). Paraffin embedded lung tissue was incubated with ab2612 (1:100 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab2612 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

Immunohistochemistry (PFA perfusion fixed frozen sections) - BRIT1 antibody (ab2612)

Immunohistochemistical detection of BRIT1 using antibody (ab2612) on paraformaldehyde perfusion fixed mouse heart tissue sections. Primary antibody diluted @ 1/300 & incubated for 18 hours @ 20°C in PBS + 0.3 % Triton X100. Secondary antibody: goat anti-rabbit conjugated to biotin (1/300).  The antibody produced a weak cytoplasmic staining in cardiac cells.

Sophie Pezet, CNRS, Paris, France

Western blot - BRIT1 antibody (ab2612)

All lanes : Anti-BRIT1 antibody (ab2612) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with BRIT1 peptide (ab13737) at 1 µg/ml
Lane 5 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate with BRIT1 peptide (ab13737) at 1 µg/ml
Lane 6 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with BRIT1 peptide (ab13737) at 1 µg/ml

Lysates/proteins at 10 µg per lane.

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

Performed under reducing conditions.

Predicted band size : 93 kDa
Observed band size : 105 kDa (why is the actual band size different from the predicted?)
Additional bands at : 55-60 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 3 minutes

Western blot - BRIT1 antibody (ab2612)

All lanes : Anti-BRIT1 antibody (ab2612) at 1 µg/ml (BLOCKED IN 5% MILK)

Lane 1 : Hela Whole Cell Lysate - Bleomycin Treated (40U/ml)
Lane 2 : Hela Whole Cell Lysate - Hydroxyurea Treated (48hr, 1uM)

Lysates/proteins at 25 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 93 kDa
Observed band size : 105 kDa (why is the actual band size different from the predicted?)
Additional bands at : 19 kDa,55 kDa,60 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 12 minutes