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Protein purification questions and answers
How can I improve protein purity without reducing yield? I encountered a problem that is quite common when trying to purify proteins: I lose significantly more protein when trying to increase purity. I am currently using Ni-NTA Magnetic Agarose beads from Qiagen, but I have tried several other types of beads, each returning similar elution profiles. In addition to changing beads, I have also tried varying conditions for the purification step as follows: Wash the beads with 20 mM imidazole bu...
ELISA Protocol Coating the appropriate antigen to microplate 1. Dilute the antigen to a final concentration of 20 μg/ml in PBS. Fill the microwells of a Nunc Maxi-Sorp Immuno Plate with 50 μL of the diluted antigen. Note: Test samples containing pure antigen are usually pipetted onto the plate at less than 2μg/ml. Antigen protein concentration should not be over 20 μg/ml as this will saturate most of the available sites on the microtitre plate. 2. Incubate at 4°C overnight or 2 h at room te...
ELISA standard curve
ELISA standard curve Standards and the standard curve 1. Make up a stock solution of 0.08 ug of protein/100 ul of PBS and store at 4 C To make the stock: - Use protein extracted from fresh hyphae that are nearly 100% immunoreactive. To determine this, run a Bradford and an ELISA assay on the samples and compare concentrations. - If Bradford and ELISA values are nearly the same, make an ELISA curve and test the values by comparing results to a known curve. - Make up 500 ul aliquots o...
Western Blot: Technique, Theory, and Trouble Shooting
Western Blot: Technique, Theory, and Trouble Shooting Western blotting is an important technique used in cell and molecular biology. Researchers can identify specific proteins from a complex mixture of proteins extracted from cells using Western Blot. A mixture of proteins is separated based on molecular weight through gel electrophoresis. The gels are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the prot...
LifeTein Dot Blot protocol
LifeTein Dot Blot protocol Dot Blot protocol Dot blot is similar to the Western blot technique. However, the proteins are spotted directly onto the membrane or paper for detecting and analyzing. This is a good technique to estimate protein concentration. Procedure 1. Prepare the nitrocellulose (NC) membrane. 2. Spot 2 µl of diluted samples onto the NC membrane and let it dry. 3.Blocking: Block the membrane in 1% BSA or non-fat milk in TBST (1 hr, Room Temperature). 4. Primary Antibody: I...
Guidelines for the storage of different types of antibody
Guidelines for the storage of different types of antibody Storage temperatures and conditions For many of our antibodies, freezing at -20 C or -80 C in small aliquots is the optimal storage condition. Aliquotting minimizes damage due to freezing and thawing, as well as contamination introduced by pipetting from a single vial multiple times. Aliquots should be no smaller than 10 µl. Upon receiving the antibody, centrifuge at 5,000 x g for 30 seconds to pull down the solution, and transfe...