1.5M tris Hcl was prepared by adding 27.23g of tribase and 40 ml of deionised water by adjusting PH 8.8 and make the volume to 75 ml and was stored at 4°c.50ml of 0.5M tris hcl was prepared by adding 6g trisbase and 60ml of deionised water at the PH 6.8Loading buffer was prepared by adding 2.5 ml glycenol ,3.55ml deionised water, 10%(w/v) sds,1.25ml o.5M tris Hcl/PH 6.8,0.2ml 0.5% (w/v) bromo phenol blue and 9.5ml total volume and was stored at room temperature.10% (w/v) SDS was prepared by adding 10g of SDS in 40ml water and was marked up to 50ml.Running buffer was prepared by adding 30.3g of tris base,10g of SDS was prepared by adding 100mg of ammonium and 144g of glycine at the PH to 8.8.10% (w/v) APS was prepared by adding 100mg of ammonium persulphate in 0.5ml of deionised water.300ml of 10- TBS was prepared by adding 3.65gm of trisbase,26.3gm of Nacl at PH 7.4 and kept at 4°c.
Preparation of Gel:
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12% of SDS polyacrylamide gel was prepared by adding 3.3ml of distilled water,4ml of 30% acrylamide mixture,2.5ml 1.5M tris Hcl at PH 8.8,0.1ml of 10% SDS,0.1M 10%ammonium persulphate and 4µl of TEMED.
Preparation of stocking Gel:
5ml of 5% stacking gel was prepared by adding 30µ ml of distilled water.0.83ml of 30% acrylamide solution and 0.63ml of 0.5M tris Hcl of PH at 6.8,0.05ml 10% SDS,0.05ml of 10% ammonium persulphate and 5µl TEMED.
Glass slides were prepared to make a gel cassetle it should be arranged in right orientation and glass slides were checked with deionised water in order to avoid leakage.Then the water was discarded and 5ml of lower gel was prepared with 12% acrylamide concentration.Then 1ml of frofanol was poured to avoid air bubbles and then the gel was left for 30-40 minutes.Then 5ml of stacking gel was poured on the lower gel and a comb was placed on the top of the stacking gel was left for 30-40 minutes for solidification.
Samples were prepared by adding 3:1 ratio i.e. three parts of sample and one part of loading buffer and 1µl of DTT was added into the effendorf vials.Then the vials were centrifuged for 5 minutes at a maximum speed of 10000 rpm and 10 µl of molecular marks was taken .Then the samples and molecular marks was put on the hot place at 100°c for protein denaturation.Then it was placed in the cold water before loading into the wells.
The comb that was placed on stacking gel was removed from the gel.Then the gel sandwich was removed from the cassetle and was placed on the one side of the gel supporting frame.Then the wells were washed with the running buffer before loading the samples.Then half of the gel tank was filled with running buffer then molecular marks and samples were loaded into the wells.Then the rest of the tank was filled with running buffer to a certain level and lid was placed on it and was connected to the electric power supply at 200 V. Then the gel was run with constant current till the samples were moved up to ¾ th of the gel.
Membranes,(PVDF or nitro cellulose) was prepared and soaked in methanol for 15 seconds.Then it was removed from methanol and soaked in water for 2 minutes.Two blotting pads were taken which are of the same sizes of the membrane and was soaked in the transfer buffer for 15 minutes and the membrane was arranged in the sequential order i.e. first the filter paper was placed on the plate then the gel was placed on the top of the filter paper then the membrane was placed on the gel. Then the filter paper was placed on the membrane then the bay was placed on the filter paper.Then the membrane was removed carefully by using the forceps and was placed on the top of the blotting paper.Then the gel tank was filled with transfer buffer and put the transfer unit into the transfer buffer and a small magnetic stirrer was placed into the tank, then the ice block was placed in the chamber to avoid unit heating and the unit was placed in the magnetic stirrer then it was connected to the power supply at 30mA.After transferring the power supply was switched off, gel and the membrane was removed.Then the gel was stained with comassie blue for 5-10 minutes then it was kept in the destaining solution for 15 minutes each on the shaker then the protein bands were transferred from the gel into the membrane then the destaining solution was drain out and gel was kept in the fridge. Then tha PVDF membrane was marked at the upper left cornerThe membrane was stained with ponceau solution for few minutes which was on the shaker until the bands were seen on the membrane. Then the ponceau solution was put back into the bottle and the membrane was washed with deionised H2o.
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Then the protein building sites that were not occupied on the PVDF membrane was blocked with 1% milk in TBS on the shaker for 15-20 minutes.Then the blocking buffer was discarded. Then the primary antibody was prepared by the dilution of it in 1-TBS with a minimum usage of antibody.It was placed on shaker for 2 hours at room temperature then the primary antibody was poured out and the membrane was washed with 1-TBS.Then the secondary antibody was prepared by the dilution of it in 1-TBS for each antibody.The secondary antibody was incubated for 45-60 minutes with PVDF membrane at room temperature on the shaker.Then the secondary antibody was poures and was washed with 1-TBS for 5 minutes.
Preparation and detection:
The bench has been wet and the cling film placed on the bench tightly to avoid the formation of air bubbles.Then the cling film was placed on the membrane and excess water was blotted but the membrane should not be dried.
First day of immunohistochemistry
The slides were washed with TBS one time for 5 minutes. Then the slide was washed with 0.3% H2O2 in methanol and was again washed with TBS thrice for 5 minutes and was with grease pen.Then it was blocked with milk which contained 0.5g of milk,10µl of triton and 10ml of TBS for an hour.Then 1° Ab was prepared which contained 1µl Ab and 499µl of TBS and its concentration was made up to 1:500.Then the slides were placed in the humidified chamber for a night.
The milk was removed with 1°Ab and washed thrice with TBS for 5 minutes. It was blocked with milk 0.5g and 10 ml of TBS for 30 minutes. 2°Ab was prepared and diluted with 50λ Ab +50 λ TBS 2 drops of 2°Ab was put on each solution and was placed on bench for 1 hour.
the slide was washed thrice for 5 minutes with TBS.DAB was prepared by making up the concentrations of 1% nickel sulphate, 1% cobalt chloride/ nickel 300λ cobalt 300λ,0.3% H2O2 20-+100TBS,DAB 50λ+50λ TBS.All these were mixed and 2-3 drops were put on each slide. The reaction was watched and it should be left for at least 25 minutes. The slide was washed thrice with TBS for 5 minutes and left on the bench for overnight. The slides were dipped in water for 1 minute. Again the slides were dipped in alcohol in a series up to 50%, 70%, 80%, 90%, 95% and 100% and again the slides were dipped for 10 minutes in histoclear. The slides were placed on the paper towel and 2 drops of DPX was placed on each section. Then it was covered with cover slips and the slides were left to dry for overnight.
When the mice was treated with MPTP the CDK5 activity was more in substantia nigra compacta, compared to the controlled mice. By treating the mice with flavofiridol are observed that there was no increase in substantia nigra by this we can assume the activity of the CDK5 is reduced by flavofiridol due to which the loss of neurons does not occur. By using the above techniques Western blotting and Immunohistochemistry we can identify the protein level and its activity in the substantia nigra compacta.