Anti-Sall4 antibody (ab31968)

Immunocytochemistry/ Immunofluorescence - Sall4 antibody (ab31968)

ICC/IF image of ab31968 stained mouse embryonic stem cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab31968, 1µg/ml) overnight at +4ºC. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue).

Western blot - Sall4 antibody (ab31968)

Anti-Sall4 antibody (ab31968) at 1 µg/ml + E14tG2a (Mouse embryonic stem cell line) Whole Cell Lysate at 20 µg

Secondary
IR Dye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

Performed under reducing conditions.

Predicted band size : 113, 66 kDa
Observed band size : 85 kDa (why is the actual band size different from the predicted?)


This antibody detects a single band which we believe corresponds to Sall4b (Swissprot: Q6S7E9). The reason for the antibody failing to detect a band corresponding to Sall4a is unclear, although there is an extremely faint band at 140 kDa that may correspond to Sall4a. Ma et al, 2006 (PMID: 16763212) describes isoforms of Sall4 running significantly higher than would be predicted in SDS-PAGE (165 kDa and 95 kDa for Sall4a and Sall4b in human tissue, as opposed to predicted sizes of 113 kDa and 66 kDa). An alternative Sall4 antibody which detects both Sall4a and Sall4b is available as ab29112.

Western blot - Sall4 antibody (ab31968)

All lanes : Anti-Sall4 antibody (ab31968) at 1/1000 dilution

Lane 1 : Mouse ES Cell Lysate
Lane 2 : Mouse ES Cell Lysate
Lane 3 : Mouse ES Cell Lysate

Lysates/proteins at 14 µg per lane.


Predicted band size : 113, 66 kDa
Observed band size : 145 kDa (why is the actual band size different from the predicted?)
Additional bands at : 110 kDa (possible isoform,non-specific binding),70 kDa (possible isoform,non-specific binding),85 kDa (possible isoform,non-specific binding).

The image shows detection of a Sall4 band at 145 kDa. Sall4 is known to run at a higher molecular weight than predicted by its primary amino acid sequence. The lower molecular weight bands may represent isoforms or non-specific binding.

S. Rao, Harvard University