I am studying HeLa cell lysates. I determined the concentration by Bradford assay. I prepared the SDS-PAGE stacking (5%) gel and the separating gel (10%), ran the gel with Cruz molecular weight marker and 50mcg of my lysate sample at 18mA constant current and a voltage of 500V.
Next, I setup the transfer in Tris-glycine-methanol buffer for overnight at 25V constant voltage and 500mA current.
The next day I performed the blocking step with 5% casein TBS-T for 1.5hours at room temperature and next added my primary antibody [eIF4G(A-10)] at 1:1000 dilution in 5% casein TBS-T, left it overnight at 4C. NExt, washed it for 4X15min and added secondary antibody (rabbit antimouse IgG-HRP antibody in 1:2000 dilution), left it for 1.5 hours at room temperature.
I performed the detection with supersignal western pico substrate and observed the blot in UVP.
The problem is, the marker bands are slant and not so perfect and unable to see the sixth band. Also, I am unable to find my sample band.
I am uploading the pictures. The first one (I have no idea what the vertical band is!
) was with an old lysate sample. The second one is with a new lysate sample.
Please tell me where am I possibly going wrong. Thanks!