20150210 IgG Purification Ctrl, AR, LAC, MASP, HVH1

Objectives

We want to isolate IgG from patients sera. After this we’ll byotinilate them and use them for an IHQ with human nerve.

Material

• NAb Protein G Spin Kit
  • Binding buffer: 100mM phosphate, 150mM sodium chloride in 500ml ultrapure water (from the kit)
  • Elution buffer
  • Neutralization buffer
  • Columns
  • 2ml tubes
• Patients sera
  • AR
  • HVH1
  • LAC
  • MASP (1st from Miquel and 2nd from 167_17)
  • Ctrl (Xavi)
• 0.02% azide – PBS

Methods

*Centrifugations: 1min 5000G RT

1. Equilibrate all buffers and columns to RT
2. One column and one microcentrifuge tube per each sample and mark them
3. Snap off bottom closure and loosen cap on spin column. Place column in a 2mL collection tube, centrifuge for 1 minute and discard the flow-through
4. Equilibrate column by adding 400µL of Binding Buffer to the column/collection tube assembly and mix briefly. Centrifuge the column and discard the flow-through. Repeat this step once.
5. Dilute 50ul of sera into 450ul of binding buffer
6. Cap bottom of spin column with the included rubber cap, add 500µL of the antibody-containing dilution and cap column.
7. Incubate column at room temperature with end-over-end mixing for 10 minutes, when volumes allow mixing to occur.
8. Loosen cap and remove bottom cap. Place spin column in new collection tube and centrifuge for 1 minute. Save the flow-through as a FT0.
9. Transfer the column to a new collection tube. Wash column by adding 400µL of Binding Buffer. Mix briefly to suspend the resin and centrifuge for 1 minute. Repeat wash two additional times for a total of three washes.
10. Mark 3 tubes per sample as EL1, EL2 and EL3 and add 40µL of Neutralization Buffer to each one.
11. Add 400µL of IgG Elution Buffer to the spin column, mix gently and centrifuge for 1 minute into EL1 tube.
12. Transfer the spin column to EL2 collection tube that contains Neutralization Buffer, saving the collected solution.
13. Transfer the spin column to EL3 collection tube that contains neutralization buffer, saving the collected solution.
14. Determine which fraction(s) contain the purified antibody by measuring the relative absorbance of each fraction at 280nm (nanodrop).

To regenerate the used column for storage or re-use:

1. Add 400µL of Elution Buffer and centrifuge for 1 minute. Repeat three times.
2. Wash resin several times with Storage Solution and store column at 4°C. Do not allow the resin to become dry.

Typically, the immobilized protein column may be used up to 10 times without significant loss in binding capacity, although the actual number of effective usages may vary

Results

We obtained three elutions of each sample

Absorbance at 280nm: mg/ml

AR.0 – 2.7

AR.1 – 0.36

AR.2 – 0.36

AR.3 – 0.35

Ctrl.0 – 3.13

Ctrl.1 – 0.21

Ctrl.2 – 0.21

Ctrl.3 – 0.09

HVH1.0 – 2.59

HVH1.1 – 0.17

HVH1.2 – 0.23

HVH1.3 – 0.16

LAC.0 – 3.72

LAC.1 – 0.22

LAC.2 – 0.4

LAC.3 – 0.26

MASP.0 – 2.27

MASP.1 – 0.07

MASP.2 – 0.04

MASP.3 – 0.01

Results of 12.02.2015 (MASP 167_17)

FT0: 2.25

W1: 0.22

W2: 0.05

W3: 0.00

EL1: -0.07

EL2: -0.02

EL3: -0.10

Conclusions

We have to test each sample by ICC.

We’ve done ICC-CNTN for MASP.0, MASP.1, HVH1.0 and HVH1.1 and the only positive mark is for HVH1.1. HVH1.0 is weak but positive also. The two samples of MASP are clearly negative, therefore there is a problem with this sera.

*ICC methods: HEK cells transfected with CNTN. 1h blocking buffer, 1,5h samples (dilutions: nº1 1/2 and nº0 1/4), and 1h of secondary antibodies (GAH594+GAM488)

Fina will do an ICC of the others samples (13.02.2015).