Here's my protocol:
Acrylamide Gel: 10%
2.5mls 1.5m tris ph 8.8
3.33mls acrlyamide bis 29:1 ratio
100ul 10% SDS
50ul 10%APS made up on the day.
Topped off with layer of Isopropanol.
Waited 40 minutes for gel to definitely set.
Poured off the isopropanol, and gently washed out with water.
1.25mls 1mM Tris ph 6.8
650ul of Acrylamide bis 29:1 ratio
50ul 10% SDS
Waited again for 40 minutes for gel to set.
Sample loaded consisted of:
11ul of Cell lysate sample
4ul of Sample buffer (taken from 1ml of 5x Buffer with 125ul of mercaptoethanol added)
5ul of distilled water.
(only 1 sample was loaded, and a ladder was loaded too)
The sample was run at 80volts for 20 minutes before being increased to 100volts until the sample buffer appeared to be at the bottom of the gel and the ladded was visible.
It was at this point I thought there could be a problem, as the sample seemed to diffuse a little out from the well and appear like a large spot stretching out over 2 wells.
Nonetheless, I still continued with transferring (for 2 hours at 80V).
Afterwards, a ponceau stain showed there was something present.
I blocked the nitrocellulose in 5% milk powder in TBS-Tween for 1 hour.
Then I washed in TBS-T for about 5 minutes.
The primary antibody ( 1:1000 dilution of a Myosin Light Chain 2 antibody in 5%BSA in TBS-Tween) was added and the sample was left overnight shaking at 4C.
Next day, the membrane was washed 3 times for 5 minutes each time in TBS-Tween.
The Secondary antibody (an anti-rabbit antibody diluted 1:1000 in 5% BSA in TBS-Tween) was then added and the sample was left shaking at room temperature for 1 hour.
The membrane was washed again 3 times for 5-10 minutes each time in TBS-Tween.
The membrane was then developed in Pico stain and being placed in s-ray film for approximately 5-10 minutes.
Yet, nothing appeared on the x-ray film.
This protocol has worked in the past for other people in the lab, and all the developing stuff got results on the same day for other people so it's not faulty equipment that's hindering me.
Does anyone have any suggestions of where i could be going wrong? I would really appreciate some help, as i've tried altering the protocol in different ways yet still have no result. It's getting really frustrating....