Ligations, Plating, Western Blots and Taqman continued

27/11/09 -

A transformation for each ligation was set up as before, again using DHSalpha bacteria. Once plated the dishes were incubated at 37 degrees Celsius.

30/11/09 -

A western blot was run to test the quality of the Zmpste24 antibody. Two gels were used together, a lower gel and stack gel, to create a 10% acrylamide gel. The gel was made as follows:

Lower Gel:

- 3 ml acrylamide

- 2.5 ml resolving buffer (TRIS-SDS, pH 8.8)

- 4.5 ml water

- 50 microL APS

- 6.8 microL TEMED (a catalyst)

The lower gel was levelled using isopropenol which does not mix with the gel but will sit on top.

Once the gel has set pour off isopropenol.

Stacking Gel:

- 0.65 ml acrylamide

- 1.25 ml resolving buffer (pH6.8)

- 3 ml water

- 25 microL APS

- 5 microL TEMED

Pour over lower gel and leave to set.

The protein concentration of P.29 and P.30 was measured at approximately 2 microG/microL using a BIO-RAD protein assay which had been diluted 1 in 5 ( 200 microL BIO-RAD in 800 microL water).

25 microL of each protein (equal to 50 microG of protein) was loaded onto the gel along with 5 microL loading dye. A 10 microL sample from SW1353 cells was also loaded. The gel was run at 150V for 1 hour.

Once the gel had finished running the proteins were transferred to a PDVF membrane. This was done as follows:

- Wash membrane in methanol (be very careful to not touch membrane, even with gloved hands, use tweezers)

- Wash in transfer buffer to remove methanol

- Soak filters in transfer buffer

- Transfer one filter onto transfer blot

- Carefully place membrane on top of filter

- Place gel (with stack gel cut off) on top of membrane

- Place another soaked filter on top of the gel

- Place electrode over the top

- Run at 15v for 30 minutes

Once gel has run it needs to be stained with the primary and secondary antibodies. The primary antibody is specific for Zmpste24 and will stick to it on the membrane. The secondary antibody is specific for the primary antibody and will anneal to it. On the secondary antibody is a bioluminescent which will allow for the visualisation of the Zmpste24 protein on the membrane.

When the gel has finished running block in 6% milk (3g Marvel in PBS Tween, a buffer with detergent in it) for 1 hour.

Then the primary antibody is added. The membrane was left in the cold room at 4 degrees Celsius over night.

Once again no colonies had grown on the plates with the vector containing the insert. This could be due to my pipetting technique so we decided to run one more ligation and transformation with Tracey helping me to ensure the right concentrations are used. Only one ligation was done using 10microL of insert and 1 microL vector with the same concentrations of buffer and ligase as before. The final volume was brought to 20 microL using water. The ligation was incubated at 16 degrees Celsius over night in the PCR machine.

The vector only plate did have colonies this time. This shows the ligation is working.

1/12/09 -

The membrane with the primary antibody was washed in PBS Tween twice shaking each time for 5 minutes. The secondary antibody was then applied then washed with PBS Tween twice for 5 minutes, shaking.

Another transformation was done using the ligation made the previous day as well as the DNA made from the complimentary miR-34a positive control. The same method was used as before.

When the membrane had finished washing a chemiluminescent substrate (1ml mixed with 9 ml water to give a 1 in 10 dilution) was poured over the membrane and left for 1 minute.

The excess was drained off and the membrane put in a light proof cassette. Once in the dark room an Auto-RAD sheet was put in the light proof cassette and left to expose for 1 minute then developed.

We got a good exposure after 1 minute so decided to do another one for 10 minutes, which gave an even better picture.

The picture showed a clear band for Zmpste24 at 50kDa for both MRC5 P.29 and MRC5 P.30 cells where as no band could be seen for the SW1353 cells (although this may have been because we loaded a smaller volume for these cells). It appeared that P.30 cells had a lower concetration of Zmpste24 as the band was much lighter compared to that of the P.29. This may be because the protein concentration loaded was different but it may also be because the P.30 cells contain less Zmpste24.

To see whether the protein concentration loaded was the same we will strip the antibodies used for Zmpste24 and will re-stain for GAPDH, a protein found in all cells. If the bands are the same size when staining for GAPDH it means the same amount of protein was loaded.

The Zmpste24 antibodies were stripped using 0.2M NaOH which was poured over the membrane and left shaking for 5 minutes. The membrane was then washed with water then PBS Tween shaking for 5 minutes each time. Then the membrane was blocked in 6% milk for 1 hour. Once the membrane had finished blocking the GAPDH primary antibody was added and the membrane left in the cold room, shaking, over night.

Next, I worked with Caroline to carry out a Taqman experiment. Using Taqman will allow me to determine the level at which a number of cell lines are expressing Zmpste24 and will allow me to compare between healthy and cancer cell lines.

17 cell lines were used:

Caroline reverse transcribed the RNA in each of the samples to give cDNA. But the cDNA was too concentrated to use for Taqman so it needed to b diluted 1 in 20 which was done by using 10 microL cDNA in 190 microL water. The Taqman plate was loaded as follows:

2 microL of each sample was loaded into wells A1-C10 as shown above and then made up to 10 microL with water.
Wells E1-G10 were loaded with 10 microL of each sample as shown. Wells D3-8 were loaded with an 18S standard curve and wells H3-8 were loaded with a Zmpste24 standard curve. Then each well was made up to 25 microL with Mastermix, forward primer, reverse primer, water and either the 18S probe (wells A1-D8) or the Zmpste24 probe (wells E1-H8).

Once each well had been loaded the plate was put in the PCR machine and run for 1 and a half hours.