The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
1/250 - 1/1000. Detects a band of approximately 52 kDa (predicted molecular weight: 45 kDa).
Use a concentration of 1 µg/ml.
May be involved in modulating chromatin formation and contribute to regulation of cell proliferation.
Belongs to the nucleosome assembly protein (NAP) family.
The acidic domains are probably involved in the interaction with histones.
Polyglutamylated by TTLL4, a modification that occurs exclusively on glutamate residues and results in polyglutamate chains on the gamma-carboxyl group. Some residues may also be monoglycylated but not polyglycylated due to the absence of functional TTLL10 in human.
Nucleus. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
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 Human NAP1L1 peptide (ab22417) at 1 µg/ml Lane 5 : A431 whole cell lysate with Human NAP1L1 peptide (ab22417) at 1 µg/ml Lane 6 : HEK293 whole cell lysate at 20 µg with Human NAP1L1 peptide (ab22417) at 1 µg/ml
Secondary All lanes : Alexa fluor goat polyclonal to rabbit IgG at 1/10000 dilution
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.
IHC image of ab21630 staining in human skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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.
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 +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.