TLS Online TPP Program

#Id: 4613

#Unit 13. Methods in Biology

TUNEL method uses TdT to add BrdU to fixed and permeabilized cells which can only incorporated into fragmented DNA of apoptotic cells.

TLS Online TPP Program

#Id: 4614

#Unit 13. Methods in Biology

BrdU is incorporated into the newly synthesized DNA, and is then detected with fluorescently labeled anti-BrdU antibodies

TLS Online TPP Program

#Id: 4615

#Unit 13. Methods in Biology

Caspases western blotting assay is also used to detect apoptosis

TLS Online TPP Program

#Id: 4616

#Unit 13. Methods in Biology

The total concentration of antibody in the equilibrium dialysis chamber is known, the equilibrium equation can be rewritten as:

TLS Online TPP Program

#Id: 4617

#Unit 13. Methods in Biology

 Scatchard plots are based on repeated equilibrium dialyses with a constant concentration of antibody and varying concentration of ligand. 
If all antibodies have the same affinity, then a Scatchard plot yields a straight line with a slope of Ka. The x intercept is n, the valency of the antibody, which is 2 for IgG and other divalent Igs. For IgM, which is pentameric, n  10, and for dimeric IgA, n  4. In this graph, antibody #1 has a higher affinity than antibody #2.

TLS Online TPP Program

#Id: 4618

#Unit 13. Methods in Biology

 If the antibody preparation is polyclonal and has a range of affinities, Scatchard plot yields a curved line whose slope is constantly changing. The average affinity constant K0 can be calculated by determining the value of Ka when half of the binding sites are occupied (i.e., when r  1 in this example). In this graph, antiserum #3 has a higher affinity (K0  2.4  108) than antiserum #4 (K0  1.25  108)