The Western Blotting procedure required us to cut the gel in half because it is the most significant part of the whole process. In that, the cutting serves to have one-half useful to staining in Coomassie stain. It enables the protein bands to attain visibility thus have the ability to assessing purification of the enzymatic protein as well as its specific activity.
STAINED GEL WESTERN BLOT
The two halves of the gel were loaded with same material; however, in appearance, they look different the reason behind it is that one half with protein mixtures gets denatured in the Blotting process. As a result, the electric current causes the proteins to shift from the gel attaching onto the membrane. The other half has its antibody undergo incubation, and it's blocked with TBST reducing its background in the process changing how it looks.
During the transfer step, this is what happens in the box. The Gel first undergoes electrophoresis that has proteins travel to the positive current if a charge is applied, but initially, when added to the gel it possesses a negative charge. With heat they tend to denature and move to the positive charge. Later after mixture separation the blotting process begins in the transfer box. The electric field perpendicular to the gel surface makes the proteins move towards the membrane attached. The sandwich acts as a protector as the blocking process begins and later buffering that detects the signal at the membrane that corresponds to target protein position that is seen on the film.
For the primary antibody in the experiment, it is Chicken anti-RuBisCo. It was made by having the NC incubated together with the specific primary antibody which is the chicken unto the protein of interest which happens to be RuBisCo into a 10ml blocking buffer. When we incubate the nitrocellulose with primary antibody, it later gets incubated with secondary antibody sticking to the primary antibody. For the secondary antibody in this experiment, it is (AP) Alkaline Phosphatase. The secondary antibody was made by having the NC incubated in Goat anti-Chicken antibody. The NC incubated in secondary antibody has it stick to the primary antibody producing a colour change upon reaction with substrates. The secondary antibody is linked to an enzyme called the Alkaline Phosphatase allowing us to see a band.
The Western Blot experiment visualized protein bands, one can spot RuBisCo band, and this is the results that we expected. It is by Western Blotting that enables identification of protein of interest in regards to the specificity of protein interactions of the antibody.
Upon estimation, on the size of the RuBisCo protein from our Western Blot, it would range from 60KDa following the gel loading as indicated from the SDS Page of chloroplast extracts. In the procedure of seeing the RuBisCo band there happens to be the background "noise" which happens to be the popping colour change. Western Blots gets the background colour because of its secondary antibody reacting with substrates like Nitro-Blue Tetrazolium, and 5-Bromo-4-Chloro-3'-IndolyPhosphate.However, in previous semesters the RuBisCo band on the Western Blot happened to be at 5kD and not the normal estimate of 45kD.The rise of such results would have been that in this procedure we used a higher antibody concentration that bound to the membranes. Alternatively, it could be an issue at the transfer step where we increased the washing time decreasing on the background of the bands' sizes.
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