Western Blot Diary 29th January 2025

Today is the final day of our labs and we are performing the last steps of western blot which is to perform chemiluminescence detection.

The main thing we did was perform chemiluminescence detection which was to add the enzyme substrate to allow for a colouration reaction for the target protein. we used a device known as a chemiluminescent imager to capture the emitted light, allowing us to visualise the protein bands on the membrane.

This takes time to process as it requires around 4 mins to develop the signal. We noticed thay that the pictures we received were not as clear as we had anticipeited. We deceided to use a stronger substrate first but we assumed that the buffer ph was to high so it will probably need to be adjusted

Another possible solution to our problem was maybe to use more primary antiblidy thatn secondary as it couldve been binded to

google image J then click on download to receive the photos

Try to determine the intensity of each of the bands from the photos

Select the area you want to sample by clicking on the box icon

and dragging it over the band of interest. Once the area is selected, use the Analyze menu then measure to quantify the intensity, allowing us to compare the expression levels of our target proteins.

Collect the mean value onece the intensity has been quantified,

Normalisation

Only one control band needs to be used

the normalized values will be calculated by dividing the mean intensity of our target protein by the mean intensity of a housekeeping protein

this means that we need to select a band from the controls

The calculation ti normalise is the bands the full proteins bcl-2 divided by. The value can be x100 for a percentage corresponding to the relative expression of the target protein

linear ranges was used to determine how much of the sample we needed to put into the well. we didi’t use this so it is considered a limitation in our study. this considered a future work we ciouldve done.

Planing for stats in disso

Samples used

Negative Control

Positive Control - Venetoclax is a BCL-2 Inhibitor, Not a Destroyer → It binds to BCL-2 and blocks its function, but the protein itself remains intact.

Sample 1

Sample 2

Sample 3

Sample 4

I want to compare the results from the samples against the negative control to see if there is an effect from our novel compound using statistical analysis
Make sure the data is normally distrubited with p value < 0.05 to validate our findings and ensure the reliability of the results.
If our results are significant, our data is reliable proving that the novel compound has a measurable effect on the target proteins compared to the negative control.
I will perform 4 separate unpaired t-tests, comparing each sample to the negative control.
However it is suggested that we use Bonferroni correction to adjust p values
With a small sample size, it could be a limitation as it may reduce the statistical power of our tests, potentially leading to Type II errors where we fail to detect a true effect.
However could we find papers that support our data with results as limitations of our sample:
- We can Acknowledge that our smaller sample size might limit the reliability or precision of our own results,
- and that findings from larger studies could provide stronger evidence for the patterns you observe.