Update: A collegeau of mine and I sat down and talked about the experiment from front to back and then I realized i did something really stupid. I mixed up two protocols basicly because I was doing a lot of things at the same time. Ended up boiling my protein sample at 100C after sonification, which would have caused degradation/aggregation. Which explains why I had problems with detecting G6Pase in the first place.

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Hi there,

I keep struggling with a blot and I am about to give up.

I'm assuming everything until antibody incubation went good.

I loaded 50ug of protein into a pre-cast gradient gel (5%-20%)

Ran the first 40min (until stacking) 10mA then increased to 40mA. Changed the running buffer once half way through. Then transferred overnight 30V (CV). Put the membrane in 2% BSA (filtered) blocking through the weekend. Incubated 1hr with my primary antibody (abcam G6Pase) 1:1000 (freshly prepared). Washed 3x 5min TBST. Incubated with my secondary antibody 1:4000 (antiRabbit, CST)

Washed 3x 5min. Did ECL, (incubation in the dark for 5min). read out and all I saw was (presumably) background.

See the pictures for my "results", please do you guys have any advice? I also tried incubation overnight with my primary antibody with another blot but I don't know why it won't work. Please if someone could help that would be great. thanks.