Question

3. Con A adsorbs strongly to the membrane, yet the HRP* protein binds only slightly. If HRP bound as strongly to the membrane as Con A, then the assay may not be pos- sible unless an intermediate step was done. What would that intermediate step be?

Answer 1

3. Con A adsorbs strongly to the membrane, yet the HRP* protein binds only slightly. If HRP bound as strongly to the membrane as Con A, then the assay may not be pos- sible unless an intermediate step was done. What would that intermediate step be? Answer: The intermediate step play important role to minimise the affinty of the HRP and ConA. The intermediate step to help in the production of the product and that are possibly becouse to the intermediary steps are involved. If HRP bound very tightly to the membrane, it would be difficult to use it for checking the biological activity of Con A. In the procedure described, samples are being spotted onto the membrane and subsequently HRP is allowed to interact with Con A. Now, wherever Con A is there, because of high affinity. HRP will bind tightly. When the membrane is washed, the loosely held HRP gets removed. In the next step the substrate is being added and the color change due to Enzyme (HRP) action is being detected. So, wherever ConA is present color change will be observed. However, if HRP bound tightly with the membrane, then it's removal will not be possible by washing. Hence, the whole membrane will be covered with HRP. So, when we add the substrate, the whole membrane will show color change, irrespective of the presence or absence of Con A. Hence, the detection will not done correctly. So, in such a situation, after putting the sample on membrane, we can treat the membrane with some compound such that the membranes HRP binding sites get blocked. Now, if we add HRP it will be able to bind only with Con A and not the rest of the membrane and hence the detection can be made successfully