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]
- Step1 : RAB7A only
- Step2 : Strip
- Step3 : 1st Rolony + RAB7A
- Step4-0 : Strip
- Step4 : RAB7A only
- Step5-0 : Strip
- Step5 : 1st Rolony + ACTB
- Step6-0 : Strip
- Step6 : ACTB only
- Step7-0 : Strip
- 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 |