2 replies to this topic

[lostandconfused]

I hope I'm posting this in the correct section. I am a masters student in a new lab (i.e. the first student) in a new lab where the PI shows little interest in my project other than getting it done as soon as possible. I've run SDS-Page bacterial lysates that I've made using the following protocol:

• Grow bacteria ~18 hours on LB plates, grow @ 37C
  
• The next day, add 1x PBS to the LB plates and mix with the bacteria to make a bacterial solution.
  
• Dilute the bacterial solution with 1xPBS to get 1 mL of bacterial suspensions with an OD600 ~ 0.5 and 0.35.
  
• Take 1 mL suspensions and centrifuge for 10K RPM for 5 minutes.
  
• Remove the 1xPBS and leave the pellet
  
• Add 200 uL of Sample buffer and Lysis buffer each (total of 400 uL) vortex well.
  
• Take the new solution and boil for 10 minutes and store at 4C
  
• Load 35 uL of this solution into 12% SDS-Page Gels of 1.5mm thickness.

These were the protocols outlined with little rhyme or reason and I followed them, but I would get fairly good bands when doing my SDS-Page and Western blots. After challenged with antibody, I wanted to identify certain proteins from my western blots (run with nitrocellulose), but I am not sure which step I would go about doing a Bradford or BCA assay to identify how much initial protein concentration I've been using. Any help would be greatly appreciated.

[Maxter]

I would go with Bradford assay for protein quantitation. It's pretty solid. Make sure you do a standard curve every single time you are measuring using this assay. I use BSA as the standard protein (it comes in solid form too, so you can make a solution knowing its exact concentration). But why do you care about the protein concentration? Western blotting is rather a qualitative technique (antibody binds protein or not)

[lostandconfused]

I wanted to get the protein concentration to be able to send out the proteins for mass spec sequencing. They require a particular amount of protein and I want to be sure that I am sending them enough