For sample preparation, I diluted it to 1 mg/ml first with a buffer, then again 2 fold diluted with another sample buffer.
For fixing I used 50 ml methanol, 10 ml acetic acid and 40 ml miliq water+ then 5 consecutive wash, each 5 minute+ for sensitize solution I used sodium thiosulphate 80 mg in 100 ml for 5 minutes+ then again 5 consecutive wash each for 5 min+ for staining i used silver nitrate 200 mg in 100 ml with 75 microlitre formaldehyde in 5 minute incubation+ the reaction with gel and silver nitrate solution occur for 15 minutes+ then 5 minute wash+ then finally developing solution with sodium carbonate 2g in 100 ml with 50 microlitre formaldehyde in 3 minute incubation+ the reaction occurs till band appears+for stop solution I used fixing solution
I will eagerly wait for your reply for giving me any suggestions.
Maybe you lost the protein at the reducing step...What are your denaturation and reducing conditions?... What type of protein are you working with?
@ismail Emir Akyildiz can you kindly elaborate how a protein got lost.
I am working with a fibrinolytic protein.
Ah, sorry for the reading failure of mine and misunderstanding of the case...I thought you observed the loss at the reducing step, and assumed that the protein may be aggregated during the denaturing step caused by heat or other chemicals (typically faced when working with membrane proteins). Some solubility issues after denaturation can also be troublesome after reducing treatment for the sample...
But your experiment suffers from a native-PAGE app...This could be derived from many reasons...Here are some checkpoints that need to be clarified to identify the problem...
- What was the percentage of the gel you used and both native and SDS-PAGE were conducted at the same gel properties? Is the pore size applicable to the native proteins?
- What is the MW of the protein in native conditions (any information obtained as estimated or calculated)?
- Is it a multimeric or monomeric structure and which mw you found at reducing condition?
- Did you also confirm the same situation at Comassie staining or did this just happen when silver stain was the visualization method?
- Are you able to confirm the solubilization of the protein at both non-reduced and reduced conditions...Should be ensure about the fully solubilized proteins loaded through the gel comb
- Can you share the native-PAGE protocol rather than the staining procedure? e.g. running and sample buffer compositions and voltage applied, etc....
Best....
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