my samples are very concentrated ie for 30 ug/mL i add an average of 9 uL of my protein sample add 2.5 uL sample buffer and 1 uL sample reducer and i complete to 35 uL with ultra-pure water. Now, this has worked in the past when my protein sample wasn't as concentrated (ie adding more sample and hence less water). I am assuming i am adding a lot of water but then am not sure how much to add now.
Your sample concentration is very less...30ug/ml ..its beyond detection level. Are you sure?
Basma Elbakary
Usually for western blots you want to load anywhere from 15-50 ug of total protein, so if you are saying your protein concentration is 30ug/mL that is way too low for detection. I would double check your concentration and your units.
Hello everyone thanks you all for your reply. Forgive me my protein concentration in each sample /mL is about 2000 ug. so for a 30 ug sample, i end up adding a small amount of the protein sample itself and have to dilute it a whole lot with water. what i have done in the past is dilute my sample with water till 35 uL just because that's the loading sample size my SDS gel can take. Mahesh Chandak Sebastian Schmitt
Shawn KruegerSebastian Schmitt
Thank you so much for your answer, let just make sure i understood you correctly for every 2.5 ug protein i should add 1 ul of water?
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