INIH3T3 cells were stained with the ab against Ash2 and with DAPI. Cells were fixed with 4% formaldehyde for 10' and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the Ash2 antibody (left) diluted 1:200 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.
Determination of the titer To determine the titer, an ELISA was performed using a serial dilution of the antibody against mouse Ash2. The wells were coated with the peptides used for immunisation of the rabbit. By plotting the absorbance against the antibody dilution (Figure 1), the titer of the antibody was estimated to be 1:24,000.
WB was performed on whole cell lysates from mouse fibroblastst (NIH3T3) and embryonic stem cells (E14Tg2a) with the ab at 1:1,000 (predicted size: 68 kDa). B. WB was performed on whole cell lysates from mouse neural stem cells (NS), transfected with GFP tagged Ash2, with the ab against mouse Ash2, diluted 1:500 in BSA/PBS-Tween. The molecular weight marker (in kDa) is shown on the left; the location of the endogenous Ash2 (68 kDa) and of the GFP tagged Ash2 (106 kDa) are indicated on the right.