Team:Stockholm/22 October 2010

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==Andreas==
==Andreas==
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===IgG protease activity assay===
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Continued the protocol and read the wavelength at 450 nm.
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'''Blank:''' 80 μl PBS + 100 μl SureBlue™ peroxidase substrate + 100 μl 1 M HCl.
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{|border="1" cellpadding="1" cellspacing="0"
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!colspan="14"|Sample loading
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|-
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!width="50" rowspan="2"|Extract dil.
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!Ab dil.
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!colspan="6" width="300"|1:500
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!colspan="6" width="300"|1:1000
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|-
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|width="50"| 
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!width="50"|1
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!width="50"|2
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!width="50"|3
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!width="50"|12
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|-
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!1.5x10<sup>5</sup>
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!A
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|colspan="2" align="center"|Ind. IgGp
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|colspan="2" align="center"|Ind. SOD
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|colspan="2" align="center"|Unind. IgGp
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|colspan="2" align="center"|Ind. IgGp
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|colspan="2" align="center"|Ind. SOD
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|colspan="2" align="center"|Unind. IgGp
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|-
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!1.5x10<sup>8</sup>
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!B
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|colspan="2" align="center"|Ind. IgGp
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|colspan="2" align="center"|Ind. SOD
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|colspan="2" align="center"|Unind. IgGp
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|colspan="2" align="center"|Ind. IgGp
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|colspan="2" align="center"|Ind. SOD
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|colspan="2" align="center"|Unind. IgGp
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|-
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!&ndash;
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!C
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|colspan="2" align="center"|PBS 1:500 (cont)
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|colspan="2" align="center"|PBS 1:1000 (cont)
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|align="center"|Blank
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|}
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====Results====
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{|border="1" cellpadding="1" cellspacing="0"
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!colspan="13"|A<sub>450</sub> measurements
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|-
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|width="50"|&nbsp;
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|-
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!A
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|align="center"|0.007
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|align="center"|0.006
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|align="center"|0.008
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|align="center"|0.008
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|align="center"|0.006
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|align="center"|0.007
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|align="center"|0.006
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|align="center"|0.007
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|align="center"|0.011
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|align="center"|0.011
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|align="center"|0.005
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|align="center"|0.006
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|-
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!B
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|align="center"|0.096
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|align="center"|0.099
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|align="center"|0.054
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|align="center"|0.056
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|align="center"|0.060
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|align="center"|0.062
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|align="center"|0.035
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|align="center"|0.033
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|align="center"|0.047
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|align="center"|0.049
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|align="center"|0.048
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|align="center"|0.050
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|-
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!C
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|align="center"|0.004
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|align="center"|0.006
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|align="center"|0.004
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|align="center"|0.003
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|align="center"|'''0.000'''
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|align="center"|&ndash;
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|}
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Results from the different antibody dilutions are somewhat contradictory. The 1.5x10<sup>8</sup>/1:500 samples indicate a higher IgG protolysis activity for the induced IgGp sample, followed by the uninduced IgGp and induced SOD, which would be the expected results from a functional IgG protease (IdeS). However, for the corresponding 1:1000 antibody dilution the results are almost opposite.<br />
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Furthermore, the 1.5x10<sup>5</sup> dilutions seem too low to give any recordable and reliable results.
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The experiment will be repeated with the 1.5x10<sup>8</sup> extracts and 1:500 as well as 1:250 antibody dilutions, so see if the results are reproducible.
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====Assay restart====
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Assay restarted as described above, with duplicate samples.
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{{Stockholm/Footer}}

Latest revision as of 11:13, 26 October 2010

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Contents

Andreas

IgG protease activity assay

Continued the protocol and read the wavelength at 450 nm.

Blank: 80 μl PBS + 100 μl SureBlue™ peroxidase substrate + 100 μl 1 M HCl.

Sample loading
Extract dil. Ab dil. 1:500 1:1000
  1 2 3 4 5 6 7 8 9 10 11 12
1.5x105 A Ind. IgGp Ind. SOD Unind. IgGp Ind. IgGp Ind. SOD Unind. IgGp
1.5x108 B Ind. IgGp Ind. SOD Unind. IgGp Ind. IgGp Ind. SOD Unind. IgGp
– C PBS 1:500 (cont) PBS 1:1000 (cont) Blank – – – – – – –

Results

A450 measurements
  1 2 3 4 5 6 7 8 9 10 11 12
A 0.007 0.006 0.008 0.008 0.006 0.007 0.006 0.007 0.011 0.011 0.005 0.006
B 0.096 0.099 0.054 0.056 0.060 0.062 0.035 0.033 0.047 0.049 0.048 0.050
C 0.004 0.006 0.004 0.003 0.000 – – – – – – –

Results from the different antibody dilutions are somewhat contradictory. The 1.5x108/1:500 samples indicate a higher IgG protolysis activity for the induced IgGp sample, followed by the uninduced IgGp and induced SOD, which would be the expected results from a functional IgG protease (IdeS). However, for the corresponding 1:1000 antibody dilution the results are almost opposite.
Furthermore, the 1.5x105 dilutions seem too low to give any recordable and reliable results.

The experiment will be repeated with the 1.5x108 extracts and 1:500 as well as 1:250 antibody dilutions, so see if the results are reproducible.

Assay restart

Assay restarted as described above, with duplicate samples.





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

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