1. Is this an appropriately designed experiment? explain your answer? 2. Compare

Question

1. Is this an appropriately designed experiment? explain your answer?

2. Compare lanes 3 to lane 4, what is the result of transfecting 5ug versus 3ug of YFP SC35? what is the basis for your conclusion?

You obtain the following results from an immunoblot based on the experiment above where the blot was probed with antibodies against YFP and GAPDH (a loading control). YFP-SC35 is approximately 65 kDA and GAPDH is approximately 35 kDa a n t i Y F P a n ti Immunoblot probed with anti YFP and GA antibody 250 150 100 Y F P- 5 c 3 S. Cz H P D H

Explanation / Answer

YFP-SC35 (Yellow fluorescence protein), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)

No, because there are multiple bands over there on the blot. Only specified antibody will have specified band size and fluorescence. The concentration will limit the band size also.YFP-SC35 protein shows uneven band size. It may be because of acrylamide concentration not even.   The amount of antibody can limit the band intensity up to some limit. After saturation of antigen and antibody there will no impact of extra amount of antibody on the band width/intensity. Lane 3rd and 4th have the difference of the band size. It may be because of antibody-antibody interaction. Band intensity of 65kDA and 35kDA proteins in 3rd and 4th lanes showing that the both the antibodies are interlinked. This relation was observed where band size of YFP-SC35 decreasing at same time the GAPDH band size increasing and vice-versa.

Here we observed the multiple bands those can because of following issues:-

Concentration of Primary/Secondary Antibody too High

If Concentration of primary and secondary antibody is too high then Antibody can bind non-specifically. Bands of higher and lower molecular weight may observe.

Excessive amount of lysate loaded on gel

If too much amount of lysate loaded onto a gel, antibodies can bind non-specifically to proteins of excessive abundance, can result in multiple bands.

Degradation of target protein (where the antibody will bind) which will result Multiple lower molecular weight bands because of poor handling of the lysates and samples prior to analysis and can be indicative of degradation of the sample.

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