BCH2MBC Metabolic Biochemistry and Cell Biology- Avocado Lipids

Task

Background

T cells were activated in the presence of different short-chain fatty acids (SCFAs). After analysing their metabolism with a Seahorse Extracellular Flux assay, you noticed that T cells incubated with Butyrate had a significantly different mitochondrial metabolism. To understand why this is the case, you decided to use TLC to analyse the lipid composition of the T cells incubated with Butyrate or neutral buffer (as a control). In addition to the T cell lipids, at the same time you decide to compare the lipid composition of some common lipid-rich food sources: avocado, egg yolk and butter. In order to identify the lipids you also run some control samples which contain single, known, lipids.

You prepared the samples in the table below, and then spotted them onto the origin of a TLC plate. When all samples were loaded, you incubated them with a series of solvent solutions to separate the polar from nonpolar lipids: Solvent #1 chloroform:methanol:acetic acid:water (50:30:8:3) and then Solvent 2 heptane:diethyl ether:acetic acid (70:30:2). When the solvents have finished running, the TLC plate is dried and then lipids are visualized with a single stain, the water-soluble dye amido black 10B. The dye preferentially interacts with relatively nonpolar entities and thus will associate with lipids.

Origin position

(circles at bottom of the image spotted left to right)

Sample identification

1

Fatty Acid standard (palmitic oleic acid)

2

Phospholipid standard (L-α-phosphatidylcholine)

3

Triacylglycerol standard (Glyceryl trioleate)

4

T cell lipids (incubated with Butyrate) – 10 mL

5

T cell lipids (incubated with buffer (control)) – 10 mL

6

Avocado lipids – 1 mL

7

Avocado lipids – 2 mL

8

Egg yolk lipids – 1 mL

9

Egg yolk lipids – 2 mL

10

11

Butter lipids – 1 mL

12

13

Butter lipids – 2 mL

Analysis of Thin-Layer Chromatography (TLC) Results 

First, insert the TLC result you downloaded here.

                                                                                                                         

Next, you should label your figure – indicating what sample has been loaded at each origin.

Origin position

(circles at bottom of the image spotted left to right)

Sample identification

1

Fatty Acid standard (palmitic oleic acid)

2

Phospholipid standard (L-α-phosphatidylcholine)

3

Triacylglycerol standard (Glyceryl trioleate)

4

T cell lipids (incubated with Butyrate) – 10 mL

5

T cell lipids (incubated with buffer (control)) – 10 mL

6

Avocado lipids – 1 mL

7

Avocado lipids – 2 mL

8

Egg yolk lipids – 1 mL

9

Egg yolk lipids – 2 mL

10

11

Butter lipids – 1 mL

12

13

Butter lipids – 2 mL

 
You then need to write a figure legend. Your figure legend should include:
  1. A title which clearly and succinctly describes the figure.
  2. A description of the technique and important experimental designs; this can include the assay name, any special treatments of the samples,
  3. A description of the results for each sample. This should be a single sentence describing each sample in the TLC, including any controls. 

Altogether, your figure legend. The following website has some handy tips and can be used as a guide: https://www.biosciencewriters.com/Tips-for-Writing-Outstanding-Scientific-Figure-Legends.aspx 

Once you have finished, save your word document with your name in the file. This file can now be uploaded in the Online Practical 3 assignment file for marking.

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