Hosuk:LabNotes/2013-11-6

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Movement test before after washing, stripping[edit]

Procedure[edit]

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


Result[edit]

  • 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[edit]

  • 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[edit]

    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[edit]

  • 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.


Summary[edit]
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


  • ACTB + RAB7A  : Overlapped spots from each other ?
    • Overlapped Count of ACTB with RAB7A, Count = 26 --> Overlapped with 1st Rolony : 14
    • Overlapped Count of RAB7A with ACTB, Count = 26 --> Overlapped with 1st Rolony : 15
    • There are about 10% (ACTB) or 5% (RAB7A) overlapped counting, which is not small number...those spots need to be neglected.


  • ACTB on ACTB only and ACTB on 1st Rolony + ACTB, : Overlapped spots from each other ?
    • Overlapped Count of ACTB on ACTB only with ACTB on 1st Rolony , Count = 120
    • Overlapped Count of upper result with 1st Rolony , Count = 70 --> similar to the number from ACTB with 1st Rolony, 74


  • RAB7A on RAB7A only and RAB7A on 1st Rolony + RAB7A : Overlapped spots from each other ?
    • Overlapped Count of RAB7A on RAB7A only with RAB7A on 1st Rolony , Count = 584
    • Overlapped Count of upper result with 1st Rolony , Count = 260 --> exactly same to the number from RAB7A Only
Counts[edit]
  • 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


Pictures[edit]
ACTB with 1st Rolony (Overlapped Count = 74) ACTB only (Overlapped Count = 157)
File:Exp2 DyeTest.lif Step5 1stRolony ACTB 20x 2k Cy5 ATTO488 ch00 MIP.tif composite2.png File:Exp2 DyeTest.lif Step5 1stRolony ACTB 20x 2k Cy5 ATTO488 ch01 MIP.tif composite2.png


RAB7A with 1st Rolony (Overlapped Count = 114) RAB7A only (Overlapped Count = 260)
File:Exp2 DyeTest.lif Step3 1stRolony RAB7A 20x 2k Cy5 ATTO550 ch00 MIP.tif composite2.png File:Exp2 DyeTest.lif Step4 AddRAB7AOnly 20x 2k Cy5 ATTO550 ch01 MIP.tif composite2.png


ACTB on ACTB+RAB7A (Overlapped Count = 130) RAB7A on ACTB+RAB7A (Overlapped Count = 268)
File:Exp2 DyeTest.lif Step7 AddACTB RAB7A 20x 2k ATTO488 550 ch00 MIP.tif composite2.png File:Exp2 DyeTest.lif Step7 AddACTB RAB7A 20x 2k ATTO488 550 ch01 MIP.tif composite2.png