PKA ELISA 6-01-11

2011-5-24 data <tab border="1"> 1 2 3 4 5 6 7 8 9 10 11 12 1.015 0.703 0.984 0.869 0.991 0.921 0.585 0.456 0.677 0.644 0.616 0.877 1.45 1.51 1.803 1.447 1.487 0.341 0.877 0.26 0.251 0.304 0.16 1.152 1.45 2.278 1.428 1.352 0.907 1.334 1.016 1.211 1.327 1.544 0.775 2.648

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Line 1: 0.5% milk in PBS as a blocking buffer; Line 2: 0.5% BSA/PBS as a blocking buffer; Line 3: PBS as a blocking buffer.

Ni plate coated with 2ug/ml pka (in PBS). Well 11 & 12 wells: no coating.

1,2; 3,4; 5 wells are patient serum; 6,7,8,9,10 wells are normal human serum; 11 well did not add the serum, 12 wells add the same serum same as well 1.

Problem: No difference between with PKA coating or without PKA coating (well 12)

2011-6-1 data: <tab border="1"> 1 2 3 4 5 6 7 8 9 10 11 12 0.293 0.249 0.123 0.289 0.094 0.213 0.152 0.36 0.395 0.564 0.105 0.091 0.43 0.383 0.199 0.389 0.231 0.357 0.074 0.311 0.495 0.526 0.162 0.135 0.327 0.439 0.383 0.575 0.309 0.47 0.293 0.377 0.623 0.692 0.102 0.15 0.33 0.291 0.441 0.636 0.317 0.481 0.285 0.232 0.499 0.711 0.142 0.19 0.337 0.45 0.316 0.605 0.311 0.458 0.311 0.337 0.674 0.468 0.043 0.184 0.337 0.268 0.212 0.661 0.337 0.246 0.295 0.225 0.517 0.524 0.041 0.083 0.358 0.628 0.552 0.789 0.429 0.371 0.558 0.171 0.491 0.863 0.04 0.42 0.172 0.374 0.27 0.885 0.237 0.419 0.447 0.351 0.634 0.605 0.044 0.088 </tab>

Line 1-4: 2ug/ml pka in PBS; Line 5-8: 10 ug/ml pka in PBS; Line 1,3,5,7: 5% BSA, 3% goat serum in PBST as blocking buffer; Line 2, 4,6,8 pierce superblocking buffer as blocking buffer; 11, 12 well are no coating wells.PBS as a blocking buffer. Line 1,2,5,6 1:10000 2nd Ab dilution; Line 3,4,7,8 1: 4000 2nd Ab dilution; well 1 667 1:2000 dilution;well2 667 1:500 dilution; well3 672 1:2000 dilution;well 4 672 1:500 dilution: well 5, 35 1:2000 dilution; well 6, 35 1:500 dilution: well 7, 34 1:2000 dilution; well 8, 34 1:500 dilution: well 9, 667 1:200 dilution; well 10, 34 1:200 dilution: well 11, no serum; well 12, 667 1:500 dilution:

2011-6-2 data: <tab border="1"> 1 2 3 4 5 6 7 8 9 10 11 12 0.106 0.107 0.13 0.139 0.077 0.078 0.147 0.15 0.064 0.065 0.164 0.167 0.073 0.074 0.066 0.063 0.059 0.058 0.127 0.123 0.138 0.133 0.106 0.108 0.089 0.089 0.092 0.094 0.092 0.091 0.068 0.066 0.086 0.086 0.06 0.065 0.049 0.13 0.053 0.051 0.101 0.098 0.133 0.13 0.078 0.081 0.085 0.083 0.151 0.146 0.228 0.234 0.112 0.116 0.26 0.23 0.085 0.086 0.279 0.24 0.098 0.097 0.077 0.074 0.067 0.067 0.157 0.158 0.177 0.184 0.134 0.136 0.139 0.136 0.123 0.124 0.119 0.123 0.08 0.082 0.119 0.11 0.068 0.071 0.055 0.216 0.049 0.05 0.149 0.154 0.196 0.207 0.125 0.121 0.113 0.122

</tab> Line 1-4: 5% BSA, 3% goat serum in PBST as blocking buffer; Line 5-8: pierce superblocking buffer as blocking buffer; all serum 1:2000 dilution;

a1,2 667 ; a3,4 668 ; a5 6, 669 ; a7 8, 670 ; a9 10 671 ; a11 12 672; b1 2 674; b3 4 929; b5 6 930; b 7 8 931; b9 10 932; b11 12 31; c1 2 32; c3 4 33; c5 6 34; c 7 8 35; c 9 10 39; c 11 12 pka ab; d1 control; d2 668; d3 4 control (no mouse serum); d5 6 667; d 7 8 668; d 9 10 32; d 11 12 34; from d 3 to d 12, use the 2%bsa in pbst as diluent, others use the 2%bsa, 1% mouse serum in pbst as diluent.

seems this time the signal is very low, next time need to put substrate for 3omin, this time 20 min.