Anti-PAH Antibody Picoband®

Creation of a Pah -KO mouse model. ( A ) The mouse genomic locus on chromosome 10 and the murine targeted allele after CRISPR gene editing are shown. Exon 1 contains the translation initiation codon. NCBI GeneID: 18478; RefSeq transcript: NM_008777. Exon 1 sequence is shadowed gray, the start codon (ATG) is framed, gRNA is shown as white letters on a black background, the asterisks and arrow indicate the position of PAM and G>T transition. The donor oligo for CRISPR targeting is in bold and underlined. ( B ) Sequencing genomic DNA samples of the founder 5349. The point mutation of G to T is indicated in the black boxes. ( C ) Western blot analyses to detect PAH protein in liver or kidney of wildtype (wt) or Hom (ko) mice. Solubilized proteins (60 µg) from mouse liver or kidney were fractionated and immune-blotted with an anti-PAH antibody. The PAH protein (red; expected size 50 kDa) is evident in wildtype mice (lanes 1, 3, 5 and 7) but is absent in Hom mice (2, 4, 6 and 8); lanes 1, 2, 5, 6) are from kidney; lanes 3, 4, 7, 8 are from liver. Equal loading is indicated by probing with antibody to beta-actin (green). M, molecular weight marker lane (bands not visible in this image).
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Characterization of Hom and Het Pah -KO mice for 6 months. ( A ) The Hom mice showed lower bodyweights than the Het mice throughout the study ( B ) Summary of blood Phe at various timepoints is shown (n = 7/group) ( C ) Summary of blood Tyr at various timepoints is shown (n = 7/group). The graphical data are represented as mean ± SD (Phe and Tyr levels in Hom vs Het mice p < 0.0001) (n = 7 mice/group). ( D ) Western blot analysis of liver homogenate using anti-PAH antibody is shown. Note the lack of PAH protein in the Hom mice whereas Het mice showed a corresponding band (~ 50 kDa) for mouse PAH protein. Each lane contained 135 µg protein. Beta actin was used as an internal control. Full-length blots/gels are presented in Supplementary Fig. 2. ( E ) Summary of % PAH positive hepatocytes in the Hom and Het mice are shown. The graphical data are represented as mean ± SD (** p = 0.0012). ( F ) Representative liver and kidney PAH-IHC images show lack of IHC signal in a Hom mouse whereas liver from a Het mouse exhibit strong IHC signal in hepatocytes, mostly in the centrilobular region. Renal cortical epithelial cells from a Hom mouse show no IHC signal as compared to Het mouse, where most proximal convoluted tubular epithelium exhibit strong signal.
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Analysis of Phe, Tyr and neurotransmitters in brains of Hom and Het Pah -KO mice at 6 months. Scatter plot charts show brain Phe, Tyr, serotonin, dopamine, L-DOPA, DOPAC, NE, 5-HIAA, and HVA. n = 6 Hom, 7 Het. Bars indicate group mean and the error bars indicate standard deviation (* p = 0.01, ** p = 0.003—0.007, *** p = 0.0001, **** p < 0.0001).
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Hair coat color in Hom- Pah KO mice. The difference in hair coat color is grossly visible ( A ). Hair coat of the Hom mouse is light brown whereas it is dark brown for the Het mouse. ( B ) Hair shafts from the Hom mouse contain less melanin pigment when compared to the Het mouse. H&E stain 20× ( C ) Summary of hair melanin scores is shown in the scatter plot graph. n = 7 Hom, 7 Het. Bars indicate group median score and the error bars indicate range (*** p = 0.0006).
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Western blot analysis of PAH using anti-PAH antibody (A00761-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human HepG2 whole cell lysates,
Lane 2: human CACO-2 whole cell lysates,
Lane 3: rat kidney tissue lysates,
Lane 4: rat liver tissue lysates,
Lane 5: mouse kidney tissue lysates,
Lane 6: mouse liver tissue lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-PAH antigen affinity purified polyclonal antibody (Catalog # A00761-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for PAH at approximately 45 kDa. The expected band size for PAH is at 52 kDa.

IHC analysis of PAH using anti-PAH antibody (A00761-1).
PAH was detected in a paraffin-embedded section of human liver cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-PAH Antibody (A00761-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of PAH using anti-PAH antibody (A00761-1).
PAH was detected in a paraffin-embedded section of mouse kidney tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-PAH Antibody (A00761-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of PAH using anti-PAH antibody (A00761-1).
PAH was detected in a paraffin-embedded section of rat kidney tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-PAH Antibody (A00761-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Flow Cytometry analysis of HepG2 cells using anti-PAH antibody (A00761-1).
Overlay histogram showing HepG2 cells stained with A00761-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-PAH Antibody (A00761-1, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.