GPC 2 and 3 PreLab Lecture

1
GPC 2 and 3 Pre-Lab Lecture

• Tom Gisel
• gisel_at_wisc.edu

2
(No Transcript)
3
(No Transcript)
4
GPC 2
GPC 3

• Inorganic phosphate assay
• Recovery of lysozyme
• Bradford and lysozyme activity assay

• Total protein concentration
• Bradford
• Microplate
• Phenol red assay
• Lysozyme activity assay

MIX your TUBES!
5
Bradford Assay on Microplate
6
Bradford continued
96-well Microplate
Each well 20mL sample 200mL Bradford
reagent In triplicate and using repeater pipet
7
Lysozyme activity assay
8
Activity assay continued
9
Activity assay contd

• FIRST, measure activity of the original egg white
• Dilute if necessary (dA/min lt0.1)
• Assay every fifth fraction (starting after void
  volume), until you find the range of fractions
  containing lysozyme activity
• Determine the left and right sides of the peak
  down to the baseline, and assay EVERY fraction in
  between
• Mix the cell suspension every time.
• Lag time 5 sec Rate time 20 sec
• Phosphate buffer at pH 6.3

10
Phenol red assay

• Using Spec21s with special adaptors that hold
  1.5ml square plastic cuvettes
• Assay all red fractions, as well as clear
  fractions on either side until a baseline reading
  is obtained

11
GPC-2 Logistics

• Equipment
• 5 Shimadzu specs (for lysozyme assay)
• 2 Spec21s (for phenol red) 3 stations for
  Bradford assay
• Five groups will start the lysozyme assay right
  away on the Shimadzu specs of the remaining
  groups, two will start at the phenol red assay
  and three will do the Bradford

12
GPC 3 Pre-lab

• Inorganic Phosphate assay
• Pool 20 Lysozyme activity
• Lysozyme activity assay
• Bradford assay
• Drawer check out

13
Inorganic phsphate assay

• Spec21 on your bench
• Molybdate is in 4 N sulfuric acid this can
  cause severe burning!!

14
Pool Lysozyme Fractions