C4-2B, HS-27A, PC-3, HS-5, MCF-7 (Conditioned medium); HEPG2, C4-2B, MCF-7 (Lysate)
Tested in dot blot and western blot formats.
Dot blot performance: (Figure 1)
The antibody performed according to expectations - the cell lines tested (HS-27A, HS-5, C4-2B, MCF-7 and PC-3) were probed for HPSE via qPCR and the dot blot data mostly agreed with the transcript expression.
Brief methods: Cells were cultured until 70% confluent and medium was replaced with serum-free Opti-MEM. After 48hrs, conditioned medium (CM) was collected and concentrated 50X using a 10 kDa molecular weight cut-off spin column. 50 uL of 50X CM was blotted in duplicate onto each well of dot blot apparatus for 1 hr. The membrane (nitrocellulose) was blocked for 1 hr @ RT in PBS + 0.1%Tween20 + 5% Non-fat dry milk. Anti-HPSE was diluted in blocking buffer at 1 ug/mL (1:2000) and incubated with membrane overnight at 4*C. Sheep anti-mouse HRP-conjugated secondary was added after washes at dilution 1:50000 and incubated at RT for 1 hr. ECL substrate used was SuperSignal West Dura.
Western blot performance:
Poor. I initially tested this product against cell lysates (C4-2B and MCF-7), but due to weak/lack of signal, I also tested in the positive control HEPG2 lysate.
Figure 2: For C4-2B and MCF-7, different concentrations of lysate were tested and 2 weak bands were detected after 5-minute exposure, one around 50 kDa and another around 65 kDa. That may be HPSE, but I expected stronger signal considering loading amount of 40 ug of lysate.
Figure 3: When testing HEPG2 (30 ug lsyate per lane), the results were not comparable to the data available on the website. Two bands were detected, a strong one at around 65 kDa and another at around 75 kDa.
Lysates were collected using RIPA buffer and Halt Protease inhibitor, then sonicated and quantified using BCA assay.
Pre-cast 10% Bis-tris gels were used for PAGE, running at 200 V for 50 minutes in MOPS SDS buffer. Transfer was done using Tris-glycine buffer with 10% methanol - 40 V for 5 hours inside cold room. Efficiency of transfer was measured using Ponceau Stain and Coomassie blue. Blocking of membrane was in 5% milk in PBST0.1%. Anti-HPSE at 1:1000 and 1:2000 was incubated overnight at 4*C. Developed was described above. Exposure time: 1 second.
Detailed protocol can be found in excel sheet provided.