Good afternoon. I have a problem detecting proteins on my gel after SDS PAGE. After extracting the protein, i quantified using the Lowry's method and had concentrations ranging between 2 to 3mg/ml. I then went forth to load 20 ul of the protein sample on a 10% seperating gel. I have tried coomassie staining to no avail. then i decided to try silver which is said to detect smaller amounts of proteins. I realise that at the end still i have no bands. Please help me as i need this so as to proceed to western blotting. Here is the silver staining method i used;
Fixative: 40% methanol; 10% acetic acid; 50% water
Wash Sol.: 30% ethanol in water
Reductant: 200 mg sodium thiosulfate in 1 liter water
Silver stain: 2 g silver nitrate; 200 uL formaldehyde in 1 liter of water. Wrap bottle in aluminum foil to prevent light from getting in.
Developer: 30 g sodium carbonate; 5 mg sodium thiosulfate; 500 uL formaldehyde in 1 L water
Stop Sol.: 5% acetic acid in water
Fix proteins in gel using fixative for 20 minutes (gel can be left in here overnight etc…)
Wash three times in Wash Sol. (about 20 minutes each)
Reduce in Reductant for 1 minute
Wash three times in water (about 30 seconds each)
Stain with Silver Stain for 20 minutes
Wash three times in water (about 30 seconds each)
Develop in Developer until bands become visible
Wash three times in water (about 30 seconds each)
Stop in Stop Sol. (Bands may fade if left in this solution; dry gels or save in sealed plastic bags at four degrees)
Trouble shoot on various issues from the annealing temp, water, co-factor (Mg2+), synthesis temp and expiration of enzyme
throw in a positive control to ensure all you conditions are working
Try gradient (7- 15% (min 3 kDa-500 kDa)) separating gel, you didn't mention the size of protein.
1. It will help you to detect intact molecule as well as fragments (protein purification steps or stability issues may cause fragments).
2. Before starting the experiment just check the pH of buffers.
Thank you
Hi Ndonwi Ngwa Elvis. I think that you should do a positive control to ensure all you conditions are working.
Dear Elvis,
What size are you looking at. from your explanation there is no protein on that gel, please run with an appropriate Mw protein marker and a known protein sample as control as suggested by Lubiao and share the gel here. Good Luck!
- Badges
- Science method