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.