Yaamini’s Notebook: Protein Extractions Round 2

It’s time to extract more protein.

Based on the samples Alex used for lipid analysis, I’ve picked the following samples for my next round of extractions. I will extract 10 more samples from round 1, and 20 samples from round 2. This way, I can understand how site location, eelgrass condition, and outplant duration affected protein expression.

Here are the samples I’ve already analyzed:

Sample Site Bare Eelgrass
Case Inlet (CI) O15 O07
Fidalgo Bay (FB) O47 O37
Port Gamble Bay (PG) O55 O77
Skokomish River Delta (SK) O119 O107
Willapa Bay O127 O142

Here are the samples I will analyze for my next round of extractions from the first outplant:

Sample Site Bare Eelgrass
Case Inlet (CI) O11 O01
Fidalgo Bay (FB) O64 O70
Port Gamble Bay (PG) O25 O71
Skokomish River Delta (SK) O96 O91
Willapa Bay O135 O131

And samples for the second outplant. One thing I noticed was that there was only one eelgrass sample collected for Case Inlet:

Sample Site Bare Eelgrass
Case Inlet (CI) O218 O222
Case Inlet (CI) O217 N/A
Case Inlet (CI) O219 N/A
Fidalgo Bay (FB) O267 O209
Fidalgo Bay (FB) O257 O214
Port Gamble Bay (PG) O297 O280
Port Gamble Bay (PG) O281 O292
Skokomish River Delta (SK) O182 O184
Skokomish River Delta (SK) O197 O202
Willapa Bay O151 O165
Willapa Bay O161 O174

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Yaamini’s Notebook: Skyline Test 2

I can now analyze my full .blib!

Emma sent my .blib file generated using all of my oyster samples. I uploaded it to OWL here.

Well, maybe not all my sample files. For some reason, pecanpie was unable to analyze files 8, 11, 17, 21, 22, and 23. Files 11 and 17 had bubbles in them and did not produce any usable data, so their exclusion makes sense. However, files 8, 21, 22, and 23 had data in them. I confirmed this during data collection by looking at the .raw files. Emma said she’ll ask Brian, but apparently he’s really busy right now and may not be able to solve the problem yet.

To analyze my .blib file, I opened up the Skyline document I created earlier. The settings I used in that document can be found here.

Step 1: Use my new .blib instead of my old one.

Under Peptide Settings » Library, I created a library for my new .blib.

imageimage 1image 2image 3

Step 2: Remove the heavy/light isotope distinction.

Because our data does not have any marked heavy or light signatures, we do not need this setting. Under Peptide Settings » Modifications, I unchecked all isotope label distinctions.

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My Skyline document looks like this:

image 1

Over the next week, I’ll spot check about 100 different peptides to see if Skyline correctly identified the peak signature and peak width. This will give me an error rate for the program.

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Laura’s Notebook: Avtech online!

Thanks to Steven & Doug for getting the Avtech probes online so we can 1) keep an eye on temp and pH remotely, and 2) download data!

Interested in what’s happening out here? Check out the Avtech Live Feed. There are lots of probes showing up on this site, but focus on the ones labeled “Temp #” and “Durafet #”.

We renamed probes; here are new and old names:

New Probe Name Old Probe Name
Temp 1 not previously recorded
Temp 2 not previously recorded
Temp-w 3 Gigas Temp (FNA Header 1)
Temp-w 4 not previously recorded
Durafet 1 Oly pH (FNA pH-Exp-Cont)
Durafet 2 Header Treatment
Durafet 3 Header Control

Note: Temp-w 3 will remain in the header tank until further notice. Other probes will likely be moved around a bit.

Some example reports:

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Laura’s Notebook: Minor adjustments

Checked on the spawning setup today and made some minor modifications.

The drippers I installed yesterday are 26 L/Hr (ordered from Dripworks.com – Woodpecker drip emitters, 6 GPH, #DNPC6). I tested flow rates today ad discovered some significant differences between lines (between 6-14 L/Hr).

To see how even flow rates are on just 8 lines I closed the valve leading to the bottom shelf. Here are measurements in seconds that each line filled 100 mL:

img_7499

Average flow rate was 13 L/Hr. This led me to believe that there isn’t enough back pressure to supply all lines with 16 L/Hr. And in reality, 26 L/Hr (6 GPH) is over-kill. I had ordered 8 L/Hr drippers for the larval rearing, so I swapped these drippers in and tested flow rates on all valves with all valves wide open.

Here are the measurements in seconds for each line to fill 50 mL with the 8 L/Hr drippers:

img_7500

Top shelf average flow rate = 5.25 L/Hr = 87.5 mL/min Bottom shelf average flow rate = 6.7 L/Hr 112 mL/min

With this low flow rate I noticed that the bottom shelf didn’t have any air bubbles, while the top shelf did. The buckets currently hold about 4 gallons, so this translates to full turn-over in 2.25 hrs and 2.85 on the bottom and top shelves, respectively.

According to the FAO’s manual, the “Hatchery culture of bivalves”, their target flow rate for heavily stocked tanks should exceed 25 mL/min. My animals are stocked at low densities (15 animals/bucket, ave. wet weight/animal = 7g), and flow rates are 3-6x greater then the minimum. I’m satisfied! I also considered that, with too high flow, I might risk flushing spermatids out of the buckets before females can “get” to them. Another added benefit of this lower flow rate is that temperature in buckets warmed up to ~17.5! The hatchery’s main heated line is set to 16degC. PSRF spawns their oysters at 18degC, and since Santos et al. (1993) showed that higher temperature corresponded to faster spawning in O. lurida I’d like temperatures to be as close to 18degC as possible.

Before leaving I decreased the algae dosing rate to 50 pulses/min, sprayed all mini-banjos/buckets clean with fresh water, and cleaned all the spawning buckets with vortex.

I also placed probes, making note of the location on manifold to assess differences in T & pH:

Probe Name Oly Group Manifold Location
Temp 1 SN-10 Amb pH-A North #1
Temp 2 NF-10 Amb pH-B North #8
Temp 3 n/a Header Tank
Temp 4 SN-10- low pH-A North #5
Durafet 1 NF-6 Amb pH-B South #6
Durafet 2 SN-10 low pH-A North #5
Durafet 3 K-10 Amb pH Top #8
HOBO “Tank 1” NF-10 low pH-B North #4

Also, here’s a sippet from the FAO manual that I cited for flow rate:

image

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