×
Please leave your comments or questions
 
Stanbio LiquiColor Triglycerides Procedure No. 2100
Author: Jennifer Beck, updated date: , view: 7, Q&A: 0
Tags: Fly, Drosophila, Triglyceride and Spectrophotometer

Procedure

1. Add 200 ul of PBS to tubes with 3x flies and homogenize.

2. Serial Dilute 50 ul of standard.

  • 50 ul = 2 ug/ul = Std07

  • 25 ul of Std07 + 25 ul PBS = 1 ug/ul = Std06

  • 25 ul of Std06 + 25 ul PBS = 0.5 ug/ul = Std05

  • 25 ul of Std05 + 25 ul PBS = 0.25 ug/ul = Std04

  • 25 ul of Std04 + 25 ul PBS = 0.125 ug/ul = Std03

  • 25 ul of Std03 + 25 ul PBS = 0.0625 ug/ul = Std02

  • 25 ul of Std02 + 25 ul PBS = 0.03125 ug/ul = Std01

3. Put 10 ul of Blanks (BL = PBS); Standards (Std01-07); and Sample (Sa01-40) into 96-well plate as illustrated below. Each blank/standard/sample has a technical replicate:


1

2

3

4

5

6

7

8

9

10

11

12

A

BL

Std01

Std02

Std03

Std04

Std05

Std06

Std07

Sa01

Sa02

Sa03

Sa04

B

BL

Std01

Std02

Std03

Std04

Std05

Std06

Std07

Sa01

Sa02

Sa03

Sa04

C

Sa05

Sa06

Sa07

Sa08

Sa09

Sa10

Sa11

Sa12

Sa13

Sa14

Sa15

Sa16

D

Sa05

Sa06

Sa07

Sa08

Sa09

Sa10

Sa11

Sa12

Sa13

Sa14

Sa15

Sa16

E

Sa17

Sa18

Sa19

Sa20

Sa21

Sa22

Sa23

Sa24

Sa25

Sa26

Sa27

Sa28

F

Sa17

Sa18

Sa19

Sa20

Sa21

Sa22

Sa23

Sa24

Sa25

Sa26

Sa27

Sa28

G

Sa29

Sa30

Sa31

Sa32

Sa33

Sa34

Sa35

Sa36

Sa37

Sa38

Sa39

Sa40

H

Sa29

Sa30

Sa31

Sa32

Sa33

Sa34

Sa35

Sa36

Sa37

Sa38

Sa39

Sa40

4. Add 50 ul of Activator to every 5 ml of Reagent. Invert gently 3-4 times.

  • Let sit for 15 min at room temperature before use.

  • After 15 min, add 190 ul Activator/Reagent solution to each well of plate.

    • 190 ul A/R solution + 10 ul sample = 200 ul total

  • Let plate sit at least 10 min (30 min okay) at room temperature.

5. Turn on SpectraMax M2 machine and computer.

6. Press "drawer" button to open/close plate drawer and load your plate.

7. Open SoftMax Pro software. If "no port selected" choose Com1.

  • Click 'plate' to read plate, 'cuvette' to read cuvette

  • Save as

  • Settings

    • aborbance

    • wavelength: 500 nm

    • endpoint

    • automix

    • OK

  • Template

    • unknown

    • concentration ug/ml

    • standards

    • 1st blank

    • group

    • new group setting

    • name = sample

  • Results

    • Result Interpx(Plot#1@Graph#1,Values)

    • R2 should be ~1

    • y = absorbance, x = concentration, B = slope

    • y = A+Bx => x = (y-A)/B

  • TGtotal/corrected fly weight = ug/mg = plot this value


Comment
Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.