Anti-Haptoglobin/HP Antibody Picoband®

Western blot analysis of Haptoglobin/HP using anti-Haptoglobin/HP antibody (PA1599).
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 placenta tissue lysates,
Lane 2: human HepG2 whole cell 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-Haptoglobin/HP antigen affinity purified polyclonal antibody (Catalog # PA1599) 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 Haptoglobin/HP at approximately 45 kDa. The expected band size for Haptoglobin/HP is at 42 kDa.

Anti-Haptoglobin antibody, PA1599, IHC(P)
IHC(P): Human Lung Cancer Tissue

Effects of sample fixation on the retention of proteins of the method using PVDF membranes. ( a ) Indicated numerals are amounts (5.0, 2.5, 1.3, and 0.6 μg) of the pooled serum proteins were subjected to 10% SDS-PAGE. The blotted membranes were treated using the traditional (left panel) or optimised fixation protocol (right). ( b ) Staining intensities were statistically analyzed (n = 3 individual experiments). Solid bar, no fixation; White bar, optimised fixation protocol. The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. * Significantly different p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All values are means ± S.E. (error bars).
Index in PubMed under a CC BY license. PMID: 31040299

Fixation-dependent differences in lectin staining intensities when using PVDF and nitrocellulose membranes. Pooled human serum proteins (3 μg) were separated on 10% SDS-PAGE, and the proteins were transferred onto PVDF and nitrocellulose membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments and followed by staining with lectins (LCA and SNA). Lane i, CBB staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, sample heating at 100 °C; lane v, organic solvent (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) treatments at room temperature; lane vi, organic solvent treatments followed by sample heating at 100 °C. All treatments were applied for 30 min. Left, WB pattern; right, quantitative analysis (n = 3 individual experiments). The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. ** Significantly different p < 0.01, *** p < 0.001, **** p < 0.0001. All values are means ± S.E. (error bars).
Index in PubMed under a CC BY license. PMID: 31040299

Fixation method-dependent differences in the immunostaining intensity. ( a ) Pooled human serum samples (10 μg) were analysed by 10% SDS-PAGE, and the separated proteins were transferred onto PVDF (top) and nitrocellulose (bottom) membranes, the whole membrane was cutted into eight pieces for subsequently fixation treatments. Representative images, showing anti-human IgG antibody staining after the following treatments: lane i, Coomassie Brilliant Blue (CBB) staining; lane ii, no fixation; lane iii, drying at room temperature; lane iv, heating at 50 °C; lane v, heating at 100 °C; lane vi, immersion into the organic solvents (acetone and 50% methanol for PVDF and nitrocellulose membranes, respectively) at room temperature; lane vii, immersion into the organic solvents at 0 °C; lane viii, immersion into the organic solvents at 0 °C followed by sample heating at 100 °C; lane ix, immersion into the organic solvents at 0 °C followed by sample heating at 50 °C. All treatments were performed for 30 min. The relative intensity was shown on the right (n = 3 individual experiments). ( b ) Proteins were separated by 10% SDS-PAGE, and transferred onto PVDF membranes, the whole membrane was cutted into five pieces for subsequently fixation treatments. Fixed in acetone at 0 °C, and heated at different temperatures for 30 min prior to the immunostaining. Lane i, CBB staining; lane ii, no fixation; lane iii, heating at 25 °C; lane iv, heating at 50 °C; lane v, heating at 75 °C; lane vi, heating at 100 °C. Right panel shows the relative intensity at different temperatures. The exposure times were the same in all procedures. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. ** Significantly different p < 0.01, *** p < 0.001, **** p < 0.0001. All values are means ± S.E. (error bars).
Index in PubMed under a CC BY license. PMID: 31040299

Sensitivity of the LB method coupled with the fixation step, when using PVDF membranes. Indicated numerals are amounts (3.0, 1.5, 0.7, 0.3, and 0.1 μg) of the pooled serum proteins were subjected to 10% SDS-PAGE. ( a ) The blotted membranes were treated using the traditional (top panel) or optimised fixation protocol (bottom panel). ( b ) Quantification of band intensities were statistically analyzed (n = 3 individual experiments). Solid bar, no fixation; White bar, sample fixation. The exposure times were the same for the same lectin blotting using fixation or no fixation. Band intensities were analysed and compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6. * Significantly different p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. All values are means ± S.E. (error bars).
Index in PubMed under a CC BY license. PMID: 31040299

Application of the optimised immunostaining and lectin staining methods. ( a ) CFTR levels in HT-29 cells. ( b ) HIF-1α levels in HEK-293T cells. ( c ) GAPDH levels in liver tissue of mouse. Various amounts (quantity represented in μg) of total cellular proteins analysed using 8% SDS-PAGE and immunostained using PVDF membrane and treated with or without fixation treatments. ( d ) AFP levels in the sera of healthy volunteers (n = 6) and HCC patients (n = 7), with different sample volumes using the PVDF membranes, with or without the fixation. ** Significantly different p < 0.01, *** p < 0.001, **** p < 0.0001. All values are means ± S.E. (error bars). ( e ) AAL and PHA-E staining, using 6 μg of proteins from the sera of healthy volunteers (n = 6) and prostate cancer patients (PC, n = 10), blotted on PVDF membranes, with or without fixation. Three representative healthy samples (lane i, ii, iii) and seven representative prostate cancer samples (lane iv-x) are presented (left). Boxplot provides the quantification of the total band intensities (right). Circle, healthy subjects; square, prostate cancer patients. Student’s t-test. ** P < 0.01 and **** P < 0.0001, healthy subjects vs. PC patients; # P < 0.0001, No fixation vs fixation groups. Band intensities were compared using Image Lab software (Bio-Rad Laboratories) and GraphPad Prism version 6.
Index in PubMed under a CC BY license. PMID: 31040299