WEEK FOUR

- MONDAY 17/7/17 - 

After discussing the western blot data with Connie, she suggested it would be a good idea to do repeats with 3G3 and also 1A3 and 1B6 at 1:5 dilution rather than 1:10.  I also used a rabbit FLAG antibody with the 3G3 as a positive control on one of the membranes.

I've also been given a new job in the lab -
I'm now in charge of refilling the pipette tip boxes, which is a lot more exciting than it sounds! :)

- TUESDAY 18/7/17 - 

Today I attempted to finish the second stage of the western blot experiment as well as run a FACS experiment to see if the Tspan14 mAbs recognised endogenous Tspan14 on other cell lines including Jurkat T cells and A549 lung epithelial cells. 

As you can imagine, trying to complete two experiments simultaneously was so stressful - especially having several timers going off at once!  

(1) WESTERN BLOT EXPERIMENT

After staining the membrane with the secondary antibody solution and several washing and incubating steps, I analysed the blot using the Odyssey scanner.  Unfortunately, the blots didn't come out as well as the previous ones, because the buffer kept leaking from the inner chamber whilst I was running the gel. 😢 

RESULTS

Even with the 1:5 dilution of the primary antibody solution, there was no band present with hTspan14.  This strongly suggests the antibodies aren't suitable to be used in western blotting.

(2) FACS EXPERIMENT 

Before starting the experiment, I did a 1:5 split of the A549 and HEK cells to keep them growing.  The rest of the cells were used in the FACS experiment.  I couldn't split the Jurkat cells, as they require a different media. 

The FACS sample prep went well, apart from the fact that the ice kept melting!!  

Running the FACS samples-(the FACS machine definitely doesn't like me!)

Connie and Ally watched as I ran the sample today - we were all still optimistic that there'd be some interesting data! (We were wrong!) The scanner had been set up to stop once 10,000 cells had been collected, however when running my second MOPC control for A549 cells, the FACS machine decided to go crazy and collected up to 30,000 cells... so I ran out of sample.  Without the control, I couldn't run the rest of the samples and will have to repeat the experiment with A549 cell line another time 😞

SUMMARY OF RESULTS

 The results show that there is no shift with any of the antibodies and the peaks overlay the MOPC control almost perfectly, which is disappointing since these cell line have relatively high surface levels of Tspan14.

- WEDNESDAY 19/7/17 -

Today I checked on my A549 and HEK cells to make sure they were growing.  I only split them yesterday, so they weren't ready for experimenting today. 

Then I spent the rest of the day reading these papers:

1) Reyat JS, Chimen M, Noy PJ, Szyroka J, Rainger GE, Tomlinson MG.

ADAM10-Interacting Tetraspanins Tspan5 and Tspan17 Regulate VE-Cadherin

Expression and Promote T Lymphocyte Transmigration. J Immunol. 2017 Jun 9. pii:

ji1600713. doi: 10.4049/jimmunol.1600713. [Epub ahead of print] PubMed PMID:

28600292.

2) Jouannet S, Saint-Pol J, Fernandez L, Nguyen V, Charrin S, Boucheix C, Brou C,

Milhiet PE, Rubinstein E. TspanC8 tetraspanins differentially regulate the

cleavage of ADAM10 substrates, Notch activation and ADAM10 membrane

compartmentalization. Cell Mol Life Sci. 2016 May;73(9):1895-915. doi:

10.1007/s00018-015-2111-z. Epub 2015 Dec 19. PubMed PMID: 26686862; PubMed

Central PMCID: PMC4819958.

- THURSDAY 20/7/17 - 

Today I repeated the FACS experiment with the A549 cells. It was strange to see a slight positive shift with the last four antibodies. After discussing these results with Connie and Ally, we doubt the shifts are significant.  However, I will do a repeat next week to confirm that this is the case. 

None of the antibodies recognise endogenous Tspan14 on A549 cells.  This result is quite disappointing as the mRNA data shows that Tspan14 is a major TspanC8 in this particular cell line. 

In the afternoon, I watched Connie freeze some cells in media containing DMSO and then thaw and plate some frozen cells from the liquid nitrogen tank. 

- FRIDAY 21/7/17 - 

I checked on my HEK cells, but they still weren't completely confluent.  So instead of doing a 1:10 split, I did a 1:5 split to use them in experiments next week.

In the afternoon, I went to the MCSH meeting.  The talk this week was about the role of ARF6.

HALF WAY THROUGH ALREADY!!!!