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Griffin:Antibody Basics: Difference between revisions
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The data demonstrate that the new protocol is robust and delivers mAB of a high purity and yield as compared to the traditionally purified mAB. The application of ultrafiltration to mAB purification will be of considerable value to any researcher interested in screening hybridoma libraries and accelerating the purification of mABs. | The data demonstrate that the new protocol is robust and delivers mAB of a high purity and yield as compared to the traditionally purified mAB. The application of ultrafiltration to mAB purification will be of considerable value to any researcher interested in screening hybridoma libraries and accelerating the purification of mABs. | ||
== | ==Validation of Specificity== | ||
Validation primary antibody specificity is an ongoing topic of the utmost importance to scientists who choose to utilize immunoglobulin-based approaches. | |||
===Peptide neutralization=== | |||
The disappearance of staining in the presence of blocking peptide, i.e. preincubation of the antigen against which the antibody was raised with the antibody in theory should saturate antigen binding fragments and thereby no staining should be observed. | |||
===Knockout animals=== | |||
Disappearance of staining in knock-out animals for the target protein is particularly useful for the validation of immunohistochemical studies. | |||
===RNAi=== | |||
Reduction of staining by knock-down approaches utlizing siRNA, shRNA, Lentivirus. Further validation of the knockdown approach would employ RT-PCR. | |||
===In Situ hybridization=== | |||
===Comparative labeling=== | |||
Comparison of staining in immunoblots or immunocytochemistry for the target protein vs. related subtypes when expressed in the same cell line. The idea that similar gene class should comparative stain similar structures in systems. | |||
===Epitope comparison=== | |||
Comparing antibodies raised against multiple distinct epitopes of a gene product may yield similar staining or banding patterns. However epitope detection may also be under the influence of steric factors (cis or trans) that could influence the hydrostatic access of the antigen. | |||
==References== | ==References== | ||
