Anti-SMN/Gemin 1 antibody [2B1]

• Flow Cyt

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Flow Cytometry - Anti-SMN/Gemin 1 antibody [2B1] (AB5831)

Overlay histogram showing HepG2 cells stained with ab5831 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab5831, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 100% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMN/Gemin 1 antibody [2B1] (AB5831)

IHC image of ab5831 staining in human normal cervical carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab5831, 5µg/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.

• ICC/IF

AbReview23998****

Immunocytochemistry/ Immunofluorescence - Anti-SMN/Gemin 1 antibody [2B1] (AB5831)

ab5831 staining Gemin 1 in human HeLa cells by Immunocytochemistry/ Immunofluorescence. The cells were fixed in methanol and then blocked using 0.2% fish scale gelatin for 1 hour at 25°C. Samples were then incubated with primary antibody at 1/300 for 20 minutes at 25°C. The secondary antibody used was a donkey anti-mouse IgG conjugated to Alexa Fluor® 488 (green) used at a 1/500 dilution. Counterstained with DAPI (blue).

Gemin 1 is clearly visible in the cytoplasm and also as small dots in the nucleus (cajal bodies).

Image courtesy of an anonymous Abreview.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SMN/Gemin 1 antibody [2B1] (AB5831)

ICC/IF image of ab5831 stained HeLa 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 (ab5831, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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) at a concentration of 1.43µM.

• WB

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Western blot - Anti-SMN/Gemin 1 antibody [2B1] (AB5831)

Gemin 1contains a number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

All lanes:

Western blot - Anti-SMN/Gemin 1 antibody [2B1] (ab5831) at 1 µg/mL

Lane 3:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 4:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) (ab65485) at 1/3000 dilution

Predicted band size: 32 kDa

Observed band size: 37 kDa,45 kDa

true

Exposure time: 20min

• WB

AbReview58470****

Western blot - Anti-SMN/Gemin 1 antibody [2B1] (AB5831)

All lanes:

Western blot - Anti-SMN/Gemin 1 antibody [2B1] (ab5831) at 1/100 dilution

All lanes:

Xenopus laevis st II-III oocytes whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG Fc (HRP) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-fc-hrp-ab97265'>ab97265</a>) at 1/25000 dilution

Predicted band size: 32 kDa

Observed band size: 31 kDa

false

Exposure time: 30s

This image is courtesy of an abreview submitted by Samantha Jeschonek.