User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/06: Difference between revisions
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==Summary== | ==Summary== | ||
===cAMP precipitation=== | |||
*Beads were washed as described [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/04/09|09April2010]] | |||
*50 μL of NuPage LDS sample buffer + DTT was added to the beads (800 μL LDS sample buffer + 200 μL 1 M DTT) | |||
** Beads were put to 70 °C for 10 min. | |||
* Samples were run on NuPage prefabricated Bis-Tris gradient (4-12%) gel in MES buffer (with 500 μL Antioxidant in inner chamber (~200 mL)). 200 V. | |||
{| border="1" | |||
|+'''Gel loading hTERT D9 + HEK293 & HeLa -S see codes [[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/06/21|21June2010]]''' | |||
! align="center" style="background:#f0f0f0;"|# | |||
! align="center" style="background:#f0f0f0;"|<u>1</u> | |||
! align="center" style="background:#f0f0f0;"|<u>2</u> | |||
! align="center" style="background:#f0f0f0;"|<u>3</u> | |||
! align="center" style="background:#f0f0f0;"|<u>4</u> | |||
! align="center" style="background:#f0f0f0;"|<u>5</u> | |||
! align="center" style="background:#f0f0f0;"|<u>6</u> | |||
! align="center" style="background:#f0f0f0;"|<u>7</u> | |||
! align="center" style="background:#f0f0f0;"|<u>8</u> | |||
! align="center" style="background:#f0f0f0;"|<u>9</u> | |||
! align="center" style="background:#f0f0f0;"|<u>10</u> | |||
! align="center" style="background:#f0f0f0;"|<u>11</u> | |||
! align="center" style="background:#f0f0f0;"|<u>12</u> | |||
! align="center" style="background:#f0f0f0;"|<u>13</u> | |||
! align="center" style="background:#f0f0f0;"|<u>14</u> | |||
! align="center" style="background:#f0f0f0;"|<u>15</u> | |||
|- | |||
!Gel<br>cAMP prec. | |||
| align="center"|X | |||
| align="center"|X | |||
| align="center"|[[Media:HiMark_protein_marker_-_Invitrogen.jpg|Invitrogen Marker]]<br> (8 μL) | |||
| align="center"|HeLa -S <br>(20 μL) | |||
| align="center"|HEK293<br>(20 μL) | |||
| align="center"|[[Media:Biorad_-_Precision_Plus_Protein_Dual_Color_marker.jpg|Biorad Marker]]<br> (8 μL) | |||
| align="center"|C1<br>(20 μL) | |||
| align="center"|C2<br>(20 μL) | |||
| align="center"|C3<br>(20 μL) | |||
| align="center"|S1<br>(20 μL) | |||
| align="center"|S2<br>(20 μL) | |||
| align="center"|S3<br>(20 μL) | |||
| align="center"|X | |||
| align="center"|X | |||
| align="center"|X | |||
|- | |||
|} | |||
* Proteins were transferred using the NuPage system (transfer buufer +10% methanol), 30 V for 1.5 h | |||
** All of the markers was transferred to blot, to check gel was Coomassie stained after blotting | |||
* cAMP precipitation Ponceau S staining | * cAMP precipitation Ponceau S staining | ||
[[Image:100706_ponceau_cAMP.jpg|500px]] | [[Image:100706_ponceau_cAMP.jpg|500px]] | ||
Revision as of 08:32, 6 July 2010
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SummarycAMP precipitation
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