After being washed three times with TBST
buffer, each membrane was incubated for 1 hour at room temperature with an alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Novus Biologicals, LLC, Littleton, CO, USA) at a dilution of 1 : 5000.
After being washed with TBST
, the membranes were incubated with proper secondary antibodies (Beyotime, Shanghai, China).
Secondary antibody was added after three times of washing with TBST
buffer and then it was incubated for 1 hour.
Then strips were incubated with goat anti-mouse IgG Fc specific conjugated to horseradish peroxidase (HP) (1:5000) for 1 h at room temperature followed by vigorous washing with TBST
and addition of chemi-luminescent substrate (Vector Lab) for HP.
The goat antirabbit IgG peroxidase conjugate (category code BA1054), prestained protein molecular weight markers (category code ARI 113), N-N'-methylene-bis-acrylamide (category code AR1161), PMSF (AR1178), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein loading buffer (AR1112), TBS (AR0193), TBST
(AR0195), phosphate-buffered saline (PBS; 10 mM, pH 7.
4) and blocked with 5% milk (or 5% casein) in TBST
for 2 h at room temperature, followed by incubation with detection antibodies (40 [micro]L of detection CUZD1 antibody from the USCN ELISA kit andl:8 dilution of CA125 antibody from R&D Systems).
A mixture containing TRITC labeled anti-PML monoclonal antibody PG-M3 and 1X TBST
at a 1:10 ratio (100 [micro]L) was used for immunofluorescence staining for PML proteins on the feathered edge of each smear.
After 4 washes in TBST
, the membranes were incubated with a secondary antibody (Santa Cruz Biotechnology, Inc.
Primary antibody dilution was prepared in TBST
containing 1% BSA and incubation was carried out at 4[degrees]C for 12hrs with shaking at intervals.
Moreover was blocked using skimmed milk (5%) and further dissolved in TBST
The PVDF membrane was subsequently soaked in TBST
(TrisBuffered Saline and Tween 20) containing 5% skim milk powder and put on a shaker at room temperature for 2 hours.
Para a imunodeteccao, a membrana era bloqueada com tampao (pH 7,6) Tris HCL 0,02M, NaCl 0,15M, Twen 0,05% (TBST
), contendo 5% de leite em po desnatado, por 12 horas em agitacao suave e, em seguida, lavada 3 vezes com TBST
(10 min cada lavagem) e incubada "overnight" com anticorpo policlonal contra HSP70 (SPA-810, StressGen, Canada), diluicao 1:1000 em TBST