Lanes 1-2 : Anti-NAP1L1 antibody (ab33076) at 1/1000 dilution
Lanes 3-4 : Anti-NAP1L1 antibody (ab33076) at 1/500 dilution
Lanes 5-6 : Anti-NAP1L1 antibody (ab33076) at 1/250 dilution

Lanes 1 & 3 & 5 : U2OS whole cell lysate
Lanes 2 & 4 & 6 : U2OS whole 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), 49 kDa (possible cross reactivity), 64 kDa (possible cross reactivity)

For each lane, U2OS cells, either treated or not treated with ionizing radiation, were scraped from a 60mm dish and added to 75µl of 2X Laemmli buffer. 20µl of these samples were loaded into each lane. ab33076 recognizes a major band of approximately 52 kDa corresponding closely in size to NAP1L1.

All lanes : Anti-NAP1L1 antibody (ab33076) at 1 µg/ml

Lane 1 : HeLa whole cell lysate
Lane 2 : A431 whole cell lysate
Lane 3 : HEK293 whole cell lysate
Lane 4 : HeLa whole cell lysate with Human NAP1L1 peptide (ab22418) at 1 µg/ml
Lane 5 : A431 whole cell lysate with Human NAP1L1 peptide (ab22418) at 1 µg/ml
Lane 6 : HEK293 whole cell lysate with Human NAP1L1 peptide (ab22418) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa fluor goat polyclonal to rabbit IgG (700) at 1/10000 dilution

Developed using the ECL technique.

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), 49 kDa (possible cross reactivity), 75 kDa (possible cross reactivity)



ab33076 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.

All lanes : Anti-NAP1L1 antibody (ab33076) at 1 µg/ml

Lane 1 : NIH/3T3 whole cell lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

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: 74 kDa (possible cross reactivity)

NAP1L1 was immunoprecipitated using 0.5mg Hek293 whole cell extract, 5µg of Rabbit polyclonal to NAP1L1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hek293 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab33076.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 52kDa: NAP1L1; 49 kDa (possible cleavage fragment,cross reactivity.

IHC image of ab33076 staining in cervix 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 ab33076, 1µg/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 image of ab33076 stained A431 cells. The cells were 100% methanol fixed (5 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 (ab33076, 1µg/ml) overnight at +4°C. 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.