https://eln.excellgen.com/index.php?action=history&feed=atom&title=PKA_ELISA PKA ELISA - Revision history 2026-10-18T03:04:27Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=PKA_ELISA&diff=1065&oldid=prev Ruili.su at 13:43, 18 June 2011 2011-06-18T13:43:05Z <p></p> <p><b>New page</b></p><div>&#039;&#039;&#039;PKA-C alpha ELISA protocol&#039;&#039;&#039;&lt;br&gt;96 well plate coating:&lt;br&gt;1.In each well, add PKA-C alpha in &lt;strike&gt;TBS&lt;/strike&gt; PBS solution 100 ul (2ug/ml);&lt;br&gt;2. 4°C overnight;&lt;br&gt;3. Discard the coating solution and wash the plate with 200 ul &lt;strike&gt;TBS&lt;/strike&gt; PBS two times;&lt;br&gt;4. Add 200 ul blocking solution and store at 4°C overnight; the plate is ready for use. <br /> <br /> &lt;br&gt;ELISA:&lt;br&gt;1. Discard the blocking solution and wash the plate &lt;strike&gt;two&lt;/strike&gt; 3 times with &lt;strike&gt;TBST&lt;/strike&gt; PBST, 200 ul/time;&lt;br&gt;2. Add 100 ul serum (1:2,000 diluted with sample diluent solution) to each well; for control add 100 ul sample diluent solution;&lt;br&gt;3. &lt;strike&gt;37°C&lt;/strike&gt;, RT for 1 hour;&lt;br&gt;4. Discard serum;&lt;br&gt;5 .Wash wells three times with wash buffer, 200ul/time;&lt;br&gt;6. Add 100 ul 2nd antibody (1:10,000 in sample diluent solution); 2nd antibody for human serum is Peroxidase labeled Gt anti-human lgG (H+L) from KPL, catalog number is HRPGTXHU.05;&lt;br&gt;7. RT, 1 hour;&lt;br&gt;8. Discard the 2nd antibody;&lt;br&gt;9. Wash wells with wash buffer 6 times, 200 ul/time, 5 min incubation;&lt;br&gt;10. Add 100 ul SureBlue Reserve ELISA Substrate from KPL, catalog number: SPL-SBR;&lt;br&gt;11. RT, 30 min;&lt;br&gt;12. Add 100 ul TMB Stop Solution from KPL, catalog number: 50-85-05;&lt;br&gt;13. Record OD&lt;sub&gt;450&lt;/sub&gt; or record OD&lt;sub&gt;450&lt;/sub&gt; and use OD&lt;sub&gt;650&lt;/sub&gt; as reference. <br /> <br /> &lt;br&gt; <br /> <br /> Note for wash: for each wash, leave the wash buffer in each well for at least 30 second, shake the plate gently several times during the washing period. Invert the plate over a sink and shake it vigorously. Drain residul buffer by tapping plate on filter paper. <br /> <br /> &lt;strike&gt;&lt;u&gt;TBS&lt;/u&gt;&lt;/strike&gt; &#039;&#039;&#039;PBS solution&#039;&#039;&#039;: &lt;strike&gt;Tris&lt;/strike&gt; Phosphate Buffered Saline,&lt;strike&gt;50 mM Tris, 150 mM NaCl, pH 7.4 &lt;/strike&gt; <br /> <br /> &#039;&#039;&#039;Blocking or sample diluent solution:&#039;&#039;&#039; 1% BSA (albumin from bovine serum, Sigma catalog number A9418-50G), &lt;strike&gt;10% glycerol&lt;/strike&gt; in &lt;strike&gt;TBS&lt;/strike&gt; PBS&lt;br&gt;<br /> <br /> &lt;br&gt;&#039;&#039;&#039;Wash buffer&#039;&#039;&#039;:&lt;strike&gt;TBST (TBS&amp;nbsp;&lt;/strike&gt; PBS containing 0.05% Tween 20)&lt;br&gt; <br /> <br /> &#039;&#039;&#039;10X TBS:&#039;&#039;&#039;&lt;br&gt;* 200 ml of ultrapure water&lt;br&gt; * 500 ml of 1 M Tris.Cl pH 7.4&lt;br&gt; * 300 ml of 5 M NaCl (or use 90 grams of NaCl crystals)&lt;br&gt;&lt;br&gt;&#039;&#039;&#039;TBST:&#039;&#039;&#039; Dilute to 1X TBS, add &#039;&#039;&#039;0.05%&#039;&#039;&#039; Tween-20 (250uL Tween-20 in 500mL 1XTBS ---&amp;gt; 1X TBST)&lt;br&gt; <br /> <br /> &#039;&#039;&#039;Carbonate Coating Buffer:&#039;&#039;&#039;&lt;br&gt;3.03 g Na2CO3,&lt;br&gt;6.0 g NaHCO3&lt;br&gt;1000 ml distilled water&lt;br&gt;pH 9.6,&lt;br&gt;<br /> <br /> &#039;&#039;&#039;Stop Solution:&#039;&#039;&#039; 2M Sulfuric Acid &lt;br&gt; <br /> <br /> [[Category:Protocols]]</div> Ruili.su