IHC image of Sall4 antibody staining in a section of formalin-fixed paraffin-embedded human testis seminoma* performed on a Leica BOND^TM system using the standard protocol. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab29112, 1ug/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ICC/IF image of ab29112 stained human embryonic stem cells. The cells were fixed in Paraformaldehyde, permeabilized using 0.1% Triton X-100, blocked with 10% Goat serum, 0.1% BSA in PBS for 1 hour at RT, before incubation with ab29112 at a 1/100 dilution for 12 hours at 4°C. The secondary used was an Alexa Fluor^® 488 conjugated goat anti-rabbit polyclonal, at 1/200 dilution.

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All lanes : Anti-Sall4 antibody (ab29112) at 1 µg/ml

Lane 1 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 2 : E14Tg2a (Mouse embryonic stem cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 66, 113 kDa
Observed band size: 140,80 kDa why is the actual band size different from the predicted?
Additional bands at: 60 kDa (possible non-specific binding)


Exposure time: 8 minutes

MEF1 whole cell lysate was included as a negative control. 

 

Secondary antibody - goat anti-rabbit HRP (ab97051)

IHC-P image of Sall4 staining with ab29112 on tissue sections from a 3 year old adult marmoset testis. The sections were subjected to heat-mediated antigen retrieval using Dako antigen retrieval solution. The sections were then blocked with 5% milk for 30 minutes at 25°C, before incubation with ab29112 (1/100 dilution) for 20 hours at 4°C. The secondary was an Alexa Fluor^® 555 conjugated goat anti-rabbit polyclonal, used at a 1/500 dilution.

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Anti-Sall4 antibody (ab29112) at 1 µg + 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: 66, 113 kDa
Observed band size: 140,85 kDa why is the actual band size different from the predicted?



This antibody detects two bands which we believe correspond to the two isoforms of Sall4 outlined in Ma et al, 2006 (PMID: 16763212). This paper describes human isoforms running at 165 kDa and 95 kDa. In mouse embryonic stem cells we see both isoforms running at slightly lower molecular weights. We believe that these bands represent Sall4a (Swissprot: Q8BX22) and Sall4b (Swissprot: Q6S7E9) which are predicted to be 113 kDa and 66 kDa respectively.

ICC/IF image of ab29112 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 (ab29112, 1µg/ml) overnight at +4°C. 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).