User:Mark X. Ling/Notebook/Micb 323/2009/03/24

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Lab 9: ELISA of TNFa, and PAGE of iNOS immunoprecipitation and Western transfer

ELISA

  • dilution of TNFa standard: 118ng/mL~> 4ng/mL => 17uL stock + 484.5uL dH2O => total volume = 501.5uL
  • 1st incubation(room temperature)[1hr]@1:20->2:18
  • incubation after loading sample [1.5hr]@2:55-3:58
  • incubation after HRP [30mins]@4:02
  • only triplet was done for the supplied 8 hr LPS+IFNg
    • there were not enough sample for quadruplets
    • F12 is left empty
  • additional 50uL of concentrated strep. avidin HRP added
    • 100uL initially added = too low of concentration, so we needed more.
  • Immunoplate Grid Map
' TNFa standard Supernant Supernant
Row column 1-4 column 5-8 column 9-12
A 0pg TNFa 0hr PBS 0 next day PBS 13
B 62.5pg/mL TNFa 1st LPS 3 1st LPS+IFNg 15
C 125pg/mL TNFa 2nd LPS 5 2nd LPS+IFNg 17
D 250pg/mL TNFa 3rd LPS 7 3rd LPS+IFNg 19
E 500pg/mL TNFa Supplied 6hr LPS Supplied 6hr LPS+IFNg
F 1000pg/mL TNFa supplied 8hr LPS supplied 8hr LPS+IFNg
G 200pg/mL TNFa diluted next dayLPS 11 diluted next dayLPS+IFNg 23
H 4ng/mL TNFa 3rd IFNg 9 next day IFNg 21
    • the # indicates the sample number labeled on tube.

ELISA result:

Standard Absorbace Sample Absorbance Sample Absorbance
0 0.129 PBS 0 0.08575 PBS 24 0.1085
62.5 pg/mL 0.141 LPS .75 0.09725 LPS + IFNg .75 0.0855
125 pg/mL 0.178 LPS 1.5 0.102 LPS + IFNg 1.5 0.09325
250 pg/mL 0.212 LPS 2.25 0.10525 LPS + IFNg 2.25 0.09925
500 pg/mL 0.272 LPS 6 0.404 LPS + IFNg 6 0.20725
1ng/mL 0.409 LPS8 0.13075 LPS + IFNg 8 0.17825
2ng/mL 0.324 LPS 24 0.2265 LPS + IFNg 24 0.263
3ng/mL 0.952 IFNg 2.25 0.081 IFNg 24 0.1085

PAGE

  • heat all samples except the MW markers
    • incubated at 1:43 at 100oC for 2 minutes
  • gel started running at 2:16->2:56
Student A lane vol. content
1 15 MW marker
2 all 24hrs LPS
3 all 48hrs LPS
4 all 72hrs LPS
5 all 25hrs IFNg
6 all 48hrs IFNg
7 all 72hrs IFNg
8 all 24hrs LPS+IFNg
9 all 48hrs LPS+IFNg
10 all 72hrs LPS+IFNg
Student B 1 all 24hrs non induced
2 all 28hrs noninduced
3 all 72hrs noninduced
4 10 48hrs LPS lysate
5 10 72hrs LPS lysate
6 10 48hrs IFNg lysate
7 10 72hrs IFNg lysate
8 10 48hrs LPS+IFNg lysate
9 10 72hrs LPS+IFNg lysate
10 15 MW marker

Western Transfer

  • no comments: all procedures done according to the manual


Post lab:

  • procedure of ELISA went very smoothly
    • there was a mistake in loading the wells, but not major enough. Keep it in mind
  • didn't need to do much for Western.
  • too little reagent was used for the ELISA, no data available for our experiment
  • NOTE: result is from ELISA done by Mrs.Hinze and Jen ==> our result is not available.