This is a quick question, for my blocking step in western blotting I used 5% skim milk. Instead of dissolving the skim milk in 1X TBST, I made a mistake and dissolved it in 1X TBST containing 5% BSA (which I normally use to dilute my antibodies). I went on and incubated the membrane overnight with primary antibody. I can't stop thinking about, I am worried, will my mistake affect my result? Please help!
I transformed my pET14b vector containing my target insert into Bl21(DE3) cells and induced my culture with IPTG. I ran an SDS-PAGE to check for the solubility, expression, and whether my protein got induced. The different fractions cellular fractions I ran on gel include total cell protein fraction and soluble cytoplasmic fraction.
My problem is I cannot locate my protein on the SDS-PAGE gel. My protein size is approximately 21.38 kilodaltons. I cannot really know how my inductions really went? Can anyone provide me with feedback please. I would greatly appreciate it.
My image organization
far left-ladder, total cell protein induced, total cell protein uninduced, soluble cytoplasmic induced, soluble cytoplasmic uninduced-far right.
Here is the image12.10.2012 SDS UBC1.jpg10.7K80 downloads
I will be starting protein expression work soon. I am currently setting up an experimental design to get started. I came across an obstacle and would love to hear your inputs about it.
I will be first transforming my pET14b plasmid (has my target insert) in BL21(DE3) RIPL cells. Now, my vector has an Ampicillin resistance marker. The cells also have their own vector that possesses an antibiotic resistance marker called Chloramphenicol. When plating should my LB-agar plates contain Ampicillin or Chloramphenicol? I personally thought it should be Ampicillin (or carbenicillin-can be used instead of Amp) so that way only the cells with my pET vector survive.
I digested using EcoRI to analyze my transformants. The vector, pCR 4Blunt-TOPO, is 3956bp (an online image shows where EcoRI cuts). My insert is approx. 460-490bp. The control insert is 800bp. I ran a gel of my digestion and its attached.
The organization of the gel is
the first 10 from the left are TOPO+my insert digestion
the second following 10 are TOPO+control insert digestion 06.26.2012 ECORI digestion of TOPO 48 FB and TC2.stp.jpg16.51K221 downloads
I want to know if the digestion worked for some atleast.