As for uncoated cIEF, focusing happens when the entire pH gradient and train of bands is migrating to the capillary outlet. In terms of using coated capillary, a two-step protocol is required due to the suppression of electroendoosotic flow (EOF), including a focusing step, and mobilization. Two ways to perform cIEF: coated or uncoated capillaries. And capillary IEF (cIEF) is the technique that can quantitatively and experimentally analyze the p I, and is widely adopted in most chemistry laboratories. Amphoteric compounds are fractionated depending on their p I along a continuous pH gradient in Isoelectric focusing (IEF), which is the most common used electrophoretic technique to determine pI.
CALCULATE PI OF PROTEIN FREE
P I can be determined by various experiments, including isoelectric focusing experiments, free flow electrophoresis, capillary electrophoresis, and in-gel electrophoresis experiments using immobilized pH gradient (IPG) strips. p I is one of important physico-chemical parameters to influence overall pharmacokinetic behavior, so that it's necessary to assess p I during drug development. Isoelectric point (p I) is the pH value at which the overall net charge of a macromolecule equals zero. Thank you in advance.Isoelectric Point Determination with cIEF Is there anybody out there with experience with this method? Did it work for you? Do you have any suggestions or do you recommend any other approach of determining antibody Kd (ELISA-based would be favored I will be doing Biacore analysis also, but that can be fishy, too). It seems to me that the method is too sensitive to even very slight variations in the treatment of the two plates and that the differences can not be reliably attributed to the amount of the retained primary Ab. In the last experiment I even got a bigger difference in slopes than when using double concentration of antigen and now I am losing my confidence in the reliability of the assay.
I lowered the concentration of the antigen twice (now down to 15 nM), but I see no improvement. I am having problems with this initial experiment, as the slopes differ by more than 10%. No more than 10% of the antibody should be retained by the plate, if it is more, you should coat less antigen and repeat the experiment. By comparing the signals from both plates, you can determine the fraction of the free antibody that is retained on the first plate (by comparing the slopes of absorbance vs. Special care is taken that all the incubation and detection steps are the same for both plates.
After incubation, antibody solutions are transferred to the second plate.
CALCULATE PI OF PROTEIN SERIES
For (b), two plates are coated with Ag and a series of Ab solutions with different concentrations is added to the wells. An initial experiment has to be performed beforehand to ascertain that: (a) the antibody concentration used is in the linear range of the ELISA response (so that the absorbance is proportional to the Ab concentration) (b) only a small fraction of the total free antibody in solution is retained on the plate (so that the measurement does not significantly affect the Ab-Ag equillibrium in solution). Basically, indirect ELISA is used to determine concentration of the free antibody in a series of solutions, pre-incubated with varying concentrations of the antigen.
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