Hello
The unbound ________ is washed off leaving only the bound antibody to the protein of interest.
Antibody
Wash cells in the tissue culture flask or dish by adding cold ________ and rocking gently. Discard PBS. (Tip: Keep tissue culture dish on ice throughout).
phosphate buffered saline (PBS)
________ is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis.
Western Blot
The _______ of the band corresponds to the amount of protein present; thus doing a standard can indicate the amount of protein present.
thickness
What is used to separate cellular debris and unwanted components during cell lysis steps.
centrifuge
Incubate for 30 minutes on ___ , and then clarify the lysate by spinning for 10 minutes at 12,000 RPM, at 4°C.
Ice
What do you pour into the gel electroporator?
running buffer
10% stacking gel is composed of H2O, 1 M Tris-HCL, 30% acrylamide/bis-acrylamide, 10 % _____, 10% APS, and TEMED
SDS (Sodium dodecyl sulfate)
Run the gel with low voltage (____) for separating gel; use higher voltage (____) for stacking gel
60V, 140V
Western blot uses two different types of agarose gel: _____ and ______ gel.
stacking and separating
The proteins when loaded on the gel have a negative _____ charge, as they have been denatured by heating, and will travel toward the positive electrode when a voltage is applied.
negative
_______ is a very important step of western blotting, as it prevents antibodies from binding to the membrane nonspecifically.
Blocking
_____ is very important as it minimized background and removes unbound antibody.
Washing
Blocking is often made with _____ or nonfat dried ____ diluted in TBST to reduce the background.
5% BSA (Bovine Serum Albumin), milk
Loading buffer contains _____ so that the samples sink easily into the wells of the gel
glycerol