PROBLEM-One or Two lanes (different samples) in blot are showing no bands randomly. Assuming any problem during work, when the gel is run again with same samples, other one/two lanes ( I mean other two samples)  show no bands, whereas the previously absent bands are detected.

 In the attached picture, it can be seen that, in the Day 1 blot, sample 1 (arrow red) was not detected, whereas in the day 2 and day 3 blot sample 1 can be seen. On the other hand sample 2 ( arrow green) was present in day 1 and day 2 blots , but not in day 3 blot and  most importantly 3rd sample (arrow blue) was present in 1st picture but not in 2nd picture and reappeared in 3rd day blot.

[ All of these blots were run in same invitrogen Surecast apparatus on different days with same set of whole cell lysates (50ug/lane), 15 kDa protein was detected with same primary and secondary antibody keeping the dilutions same each time, and were developed with Biorad Clarity ECL-Peroxide solution.]

Running condition:

15% gel was run in 25mM Tris, 190mM Glycine, 0.1% SDS buffer. The current parameter was set at 70V (constant volt) for stacking part and then it was increased up to 100 V for separation. It took 1 hour fifteen minutes to run the gel with desirable resolution.

Transfer condition:

For transfer 25mM Tris, 190mM Glycine, 20% methanol containing transfer buffer was used. For this 15kDa  protein transfer was given at constant Ampere (300mA) for 40 minutes at 4 degree Celsius.

Blocking: In 3% non fat skimmed milk in TBST for 1 hour,at room temperature and on rocker with mild speed.

Incubation with Ab: Overnight at 4 degree Celsius for primary Ab, 3 hrs at RT for secondary Ab

Excess Ab wash: With TBST (contain 0.1% Tween 20), on rocker at high speed, three times for 10 minutes each.

Development:  With Biorad Clarity ECL substrate.