Hosuk:LabNotes/2013-11-6: Difference between revisions

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*2 pixel off, 281.25nm x 2 = 562.5 nm off to the right direction  
*2 pixel off, 281.25nm x 2 = 562.5 nm off to the right direction  
[[File:Exp1_MovementTest_Before_AfterWashing_ACTB.png|600px]]
[[File:Exp1_MovementTest_Before_AfterWashing_ACTB.png|600px]]




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*#Step7-0 : Strip
*#Step7-0 : Strip
*#Step7 : ACTB + RAB7A only
*#Step7 : ACTB + RAB7A only


====Result====
====Result====
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| w/i 1st Rolony  
| w/i 1st Rolony  
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| 74  
| 74 [[Media:Exp2_DyeTest.lif_Step5_1stRolony_ACTB_20x_2k_Cy5_ATTO488_ch01_MIP.tif|Raw]]
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| 1.45
| 1.45
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| 114
| 114 [[Media:Exp2_DyeTest.lif_Step3_1stRolony_RAB7A_20x_2k_Cy5_ATTO550_ch01_MIP.tif|Raw]]
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| 2.25
| 2.25
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| 1 probe only
| 1 probe only
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| 157
| 157 [[Media:Exp2_DyeTest.lif_Step6_AddACTBOnly_20x_2k_Cy5_ATTO488_ch01_MIP.tif|Raw]]
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| 3.08
| 3.08
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| 260
| 260   [[Media:Exp2_DyeTest.lif_Step4_AddRAB7AOnly_20x_2k_Cy5_ATTO550_ch01_MIP.tif|Raw]]
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| 5.13
| 5.13
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| ACTB+RAB7A
| ACTB+RAB7A
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| 130
| 130   [[Media:Exp2_DyeTest.lif_Step7_AddACTB_RAB7A_20x_2k_ATTO488_550_ch00_MIP.tif|Raw]]
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| 2.55
| 2.55
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| 268
| 268   [[Media:Exp2_DyeTest.lif_Step7_AddACTB_RAB7A_20x_2k_ATTO488_550_ch01_MIP.tif|Raw]]
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| 5.25
| 5.25

Revision as of 17:34, 7 November 2013


Movement test before after washing, stripping

Procedure

  • Image - ACTB (ATTO550)
  • Aspirate, Add Formamide, Incubate, Aspirate, Washing with PBS
  • Image again - ACTB (ATTO550)


Result

  • 2 pixel off, 281.25nm x 2 = 562.5 nm off to the right direction

File:Exp1 MovementTest Before AfterWashing ACTB.png


ACTB, RAB7A probing test

  • 1st Rolony probe (Cy5) + ACTB (ATTO488) + RAB7A (ATTO550)  ACTB and RAB7A showed always weak signal.
  • Previous experiment (10/29/13) ACTB were seen more when added ACTB only.
  • So, let’s compare between RAB7A or ACTB and 1st Rolony probe(Cy5) together.
  • And compare to ACTB + RAB7A mix


Procedure

    1. Step1 : RAB7A only
    2. Step2 : Strip
    3. Step3 : 1st Rolony + RAB7A
    4. Step4-0 : Strip
    5. Step4 : RAB7A only
    6. Step5-0 : Strip
    7. Step5 : 1st Rolony + ACTB
    8. Step6-0 : Strip
    9. Step6 : ACTB only
    10. Step7-0 : Strip
    11. Step7 : ACTB + RAB7A only


Result

  • ACTB only had more than 1st Rolony+ACTB
  • RAB7A only had more than 1st Rolony+RAB7A
  • ACTB+RAB7A was similar to ACTB only or RAB7A only.


ACTB (%) RAB7A (%)
w/i 1st Rolony 74 Raw 1.45 114 Raw 2.25
1 probe only 157 Raw 3.08 260 Raw 5.13
ACTB+RAB7A 130 Raw 2.55 268 Raw 5.25


  • Step1 : RAB7A only,
    • RAB7A, Count = 514, Overlapped = 160


  • Step3 : 1st Rolony + RAB7A,
    • 1st Rolony Count = 5066
    • RAB7A, Count = 186, Overlapped = 114


  • Step4 : RAB7A only
    • RAB7A, Count = 591, Overlapped = 260


  • Step5 : 1st Rolony + ACTB
    • 1st Rolony Count = 5105
    • ACTB, Count = 137, Overlapped = 74


  • Step6 : ACTB only
    • ACTB, Count = 246, Overlapped = 157


  • Step7 : ACTB + RAB7A only
    • ACTB, Count = 234, Overlapped = 130
    • RAB7A, Count = 463, Overlapped = 268