Assayphile

Is it science or art?  Jennifer tells us about make tubes with lovely colors, better known as Bradford and ß-galactosidase assays, and graphs with Excel.  Guest speakers tell the PCC classes about technology transfer at OHSU and cell culture at Najit Technologies.

This week (and a half) in Portland Community College’s Bioscience Technology Program we continued work with assays, and had guest speakers in Current Topics and Cell Culture.

Last week in Lab Tech we worked on the Bradford Assay which measures the amount of protein present in a sample. (Somehow I managed to spill the cuvettes in the rack. In my defense, well, there's no defense, I had the rack upside down. I immediately quit using those trickster racks.) This week, we used the Beta-galactosidase assay to determine how much enzyme activity was present in a protein sample. Both of these assays involved making serial dilutions with components that were more difficult to pipette.  They also both required us to use the spectrophotometers and plot our data on an excel graph. From that data we then used the equation from a trendline or standard curve to determine the measurement of concentration in our unknown.  And finally, we used the R^2 number on the graph to determine how close our data points were to the best fit trendline by seeing how close the R^2 number was to 1. And let me tell you, it was so much fun! (If anyone is reading this in the Portland area, let me know if you want a lab volunteer to do assays for you during the break between fall and winter terms!)

It was near torture not to write "collaborate and listen" on the stop solution lablel for the Beta-galactosidase assay. Come to think of it. I don't know what stopped me. It was only me using the stuff. 

All of our assay work was leading up to our last lab practical which involved, (cue dramatic music here) serial dilutions, an assay and using spectrophotometer readings to create an excel graph and determine our unknowns! We were given some brief instructions telling us what needed to be assayed and what dilutions would be necessary. Using that information, we had to write up our on SOP (or lab) to complete the assignment. Our instructor warned us that this is what it will likely be like in the real world so get ready for it. My reaction? Okay! This is seriously fun. 

 

There was a bit of a curve ball thrown in when she gave us our original concentration in PPM (that’s parts per million Mom). Maybe it was only a curve ball for me. I like to calculate how much of my contributing solution I’ll need for dilutions starting from the bottom. So, for instance, if I know that the assay requires 100μL of sample to be added to reaction tubes, then I’ll use C1V1=C2V2 for my weakest dilution (adding a bit for loss during pipetting depending on the viscosity), and calculate my way up my dilutions from there. I add the answer I get for V1 to my 100μL plus pipetting margin and use that for my V2 figure for the next calculation.  So, deep in the world of serious C1V1 calculations for dilutions you can imagine my initial confusion when I got to the concentration of the chemical given and it was in PPM. Wednesday night I could not for the life of me figure out how much of my PPM monkey to use with my dilutant to end up with a 1/2.5 giraffe. That our instructor gave this to us in advance probably saved me from needing an oxygen mask during the practical. Luckily when I looked at it again on Thursday I was able to forget the PPM, leave the zoo behind, and just look at the dilution in actual parts. Phew! I saved a copy of my graph from my practical so I can post how I did. Since you won't be able to read the actual data though, here's the basics:

 

Concentration     Absorbance          Dilution

1000                       2.750               Undiluted

400                        1.179                    1/2.5

200                         0.605                   1/5 

100                         0.300                   1/10

33.33                      0.101                   1/30

13.33                      0.039                   1/75

10                           0.029                   1/100

 

 

Pretty fancy for a rookie, right? 

In Current Topics we had a guest presenter from OHSU’s Technology Transfer department come talk to us and shed some light on how OHSU takes useful findings in research and well, in my terms, pretty much broker’s the transaction. Tech transfer brokers this by facilitating a partnership connecting that research to industry specifically for the purpose of public benefit via managing intellectual property, licensing, and Material Transfer Agreements.  In some cases, OHSU may even fund a startup company.  That’s the magic that happens making it worthwhile for our government to continue funding grants for research that will eventually benefit the public.  To me it sounds a lot like escrow. I have a huge amount of respect for what they do, but I really don’t want to do it. Not now anyways. You can pretty much bet that any job involving mostly filling in the blanks on a mountain of paperwork and coordinating other people more than using a pipet and flasks or reaction tubes is probably one I will try to shy away from if there are other opportunities.  Regardless, it’s good to have an understanding of how these things work.

In cell culture we seeded six wells on three different plates at 10^4, 3x10^4, and 10^5 for cell counts at 24, 48 and 72 hours so we could graph a growth curve. Working in the multiwell plates requires some adjusted technique. (Read: I don't want to talk about the bubbles.) Actually, considering it was a first time effort, it wasn't really too bad. 

We also had a guest lecturer in Cell Culture. Beth Poore from Najit Technologies, Inc., came to talk to us about her work in cell culture. Najit is focused on making vaccines that don’t require an attenuated virus (or preparation using dairy), so they can be safely administered to the immunocompromised, elderly, exceptionally young and people with allergies to dairy.

I’ll go ahead and share a secret (well known to my classmates) about myself. I’m a huge microbiophile, virophile, immunophile,  vaccinophile… well, you get it. And yes, I am making up words.  This lecturer  had me at Hello.

While her employer’s company is still small and their lab tech is also their office manager, they’re not likely to be hiring anytime soon. That didn’t stop me from approaching her after her lecture with my card and offering to job shadow or volunteer in her lab during the break between fall and winter term.  I want to play with the toys she gets! Hell, I’ll just wash and autoclave her toys! You should have seen this wave bag they culture cells with on a rocker! (Kind of like this.) Instead of T150 flasks, she had pictures of these flask-like cell culture high rises they use too. (Ok. This is what they're really called: Corning® CellBIND® Surface CellSTACK® cell culture chambers.) It looked like cell culture Disneyland!  Disneyland I tell you!

AttachmentSize
copy_of_labpracticalgraph.pdf147.6 KB
Bio-Link Program: 
Portland Community College

You're making me miss the

You're making me miss the lab!  

The lab is a wonderful and

The lab is a wonderful and magical place. All roads lead to a lab. :)

Sometimes, I do use my

Sometimes, I do use my kitchen as a lab. smiley

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