Rat hrp conjugated anti ha clone 3f 10 antibody

For western blot analysis, 12 embryos were snap frozen at stage 11 and whole embryo protein was extracted as described in
12 (link). 50µg total protein was loaded on to pre-cast BioRad Criterion
^™ TGX 18% gels in Tris-Glycine buffer. Primary antibodies were used at 1:2000 dilution for at least 1 hour at room temperature (tubulin) or at 4°C overnight (HA): Rat HRP-conjugated anti-HA clone 3F-10 antibody (Roche; 12013819001) and mouse anti-α-tubulin clone B-5-1-2 antibody (Sigma; T5618). Anti-tubulin antibody was detected with a sheep HRP-conjugated anti-mouse antibody at 1:10000 dilution (GE Healthcare; NA931V).

For western blot analysis, 12 embryos were snap frozen at stage 12.5 and whole embryo protein was extracted as described in
12 (link). For detection of phospho-status, samples were incubated in the presence or absence of lambda protein phosphatase (NEB) for 1 hour at 30°C. 60µg total protein was loaded on to pre-cast BioRad Criterion
^™ TGX 18% gels in Tris-Glycine buffer. Primary antibodies were used at 1:2000 dilution for at least 1 hour at room temperature (tubulin) or at 4°C overnight (HA): Rat HRP-conjugated anti-HA clone 3F-10 antibody (Roche; 12013819001) and mouse anti-α-tubulin clone B-5-1-2 antibody (Sigma; T5618). Anti-tubulin antibody was detected with a sheep HRP-conjugated anti-mouse antibody at 1:10000 dilution (GE Healthcare; NA931V).