![]() Eluted sample and 20 µg of LNCaP whole cell lysate (loading control) were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with StartingBlock T20 (Product # 37543) for 1 hour. The immune complexes were captured on 50 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Antigen-antibody complexes were formed by incubating 250 µg of LNCaP whole cell lysate with 2 µg of an Androgen Receptor polyclonal antibody (Product # PA5-16750) overnight on a rocking platform at 4C. Immunoprecipitation of Androgen Receptor was performed on LNCaP cells. NOTE: The presence of Pax3 recombinant protein was determined by probing a duplicate blot with a Pax3 polyclonal antibody (Product # PA1-107), and the corresponding image is on the webpage for Product # PA1-107. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). Pax4 was detected at ~38 kD using a Pax4 polyclonal antibody (Product # PA1-108) at a dilution of 1:1000 in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugate goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for at least 30 minutes. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter (Product # 62288), and blocked with 5% milk in TBST for at least 1 hour at room temperature. Western blot analysis of Pax4 was performed by loading the indicated amounts of recombinant Pax3 (negative control) or Pax4 proteins, and 10 µL of PageRuler Prestained Protein Ladder (Product # 26616) per well onto a Novex® 4-20% Tris-Glycine polyacrylamide gel. The reaction was stopped with TMB stop solution (Product # N600) and absorbances were read on a spectrophotometer at 450-550 nm. Detection was performed using 1-Step Ultra TMB substrate (Product # 34028) for 5 minutes at room temperature. The plate was washed, then incubated with 100 µL per well of an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:5000 for 1 hour at room temperature. Wells of the plate were washed, blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543), and incubated with 100 µL per well of ovalbumin antibody (Product # PA1-196), starting at a concentration of 625 ng/mL and serially diluting 2-fold to a concentration of 2 ng/mL, for 1 hour at room temperature. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).ĭirect ELISA analysis of ovalbumin was performed by coating wells of a 96-well plate with 100 µL per well of recombinant ovalbumin protein (Product # 77120) diluted to a concentration of 3 µg/mL in carbonate/bicarbonate buffer (Product # 28382), overnight at 4C. ![]() The membrane was probed with the relevant primary and secondary antibody after blocking with 5 % skimmed milk. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). Known quantity of protein samples were electrophoresed using Novex® NuPAGE®12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). A 30 kDa band corresponding to CacyBP was observed. The blots were probed with Anti-CacyBP Rabbit Polyclonal Antibody (Product # 720326, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # 31460) at dilutions 1:10,000 (Fig. Western blot analysis was performed on whole cell extracts (30 µg lysate) of A549 (Lane 1) and Hep G2 (Lane 2).
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