Western blot - NAP1L1 antibody (ab21630)

Lanes 1 - 2 : Anti-NAP1L1 antibody (ab21630) at 1/1000 dilution
Lanes 3 - 4 : Anti-NAP1L1 antibody (ab21630) at 1/500 dilution
Lanes 5 - 6 : Anti-NAP1L1 antibody (ab21630) at 1/250 dilution

Lane 1 : U2OS cell lysate
Lane 2 : U2OS cell lysate treated with ionizing radiation
Lane 3 : U2OS cell lysate
Lane 4 : U2OS cell lysate treated with ionizing radiation
Lane 5 : U2OS cell lysate
Lane 6 : U2OS cell lysate treated with ionizing radiation


Performed under reducing conditions.

Predicted band size : 45 kDa
Observed band size : 52 kDa (why is the actual band size different from the predicted?)
Additional bands at : 49 kDa (possible cleavage fragment,cross reactivity).

For each lane, U2OS cells, either treated or not treated with ionizing radiation, were scraped from a 60 mm dish and added to 75µl of 2X Laemmli buffer. 20µl of these samples were loaded into each lane. ab21630 recognizes a major band of approximately 52 kDa corresponding closely in size to NAP1L1.

Western blot - NAP1L1 antibody (ab21630)

All lanes : Anti-NAP1L1 antibody (ab21630) at 1 µg/ml

Lane 1 : HeLa whole cell lysate at 20 µg
Lane 2 : A431 whole cell lysate at 20 µg
Lane 3 : HEK293 whole cell lysate at 20 µg
Lane 4 : HeLa whole cell lysate at 20 µg with NAP1L1 peptide (ab22417) at 1 µg/ml
Lane 5 : A431 whole cell lysate with NAP1L1 peptide (ab22417) at 1 µg/ml
Lane 6 : HEK293 whole cell lysate at 20 µg with NAP1L1 peptide (ab22417) at 1 µg/ml

Secondary
Alexa fluor goat polyclonal to rabbit IgG at 1/10000 dilution

Predicted band size : 45 kDa
Observed band size : 52 kDa (why is the actual band size different from the predicted?)
Additional bands at : 35 kDa (possible cross reactivity, but this band is not blocked),49 kDa (possible cleavage fragment,cross reactivity).

ab21630 recognizes a major band of approximately 52 kDa corresponding closely in size to NAP1L1. This band is competed away by the addition of the immunizing peptide, suggesting that this is a specific interaction.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NAP1L1 antibody (ab21630)

IHC image of ab21630 staining in human skin 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 ab21630, 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.

Immunocytochemistry/ Immunofluorescence - Anti-NAP1L1 antibody (ab21630)

ICC/IF image of ab21630 stained A431 cells. The cells were 4% formaldehyde 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 (ab21630, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.