Hosuk:LabNotes/2013-11-6: Difference between revisions
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>Hosuki78 (Created page with "*LabNote ===Movement test before after washing, stripping=== ====Procedure==== *Image - ACTB (ATTO550) *Aspirate, Add Formamide, Incubate, Aspirate, Was...") |
>Hosuki78 |
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(8 intermediate revisions by 2 users not shown) | |||
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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]] | ||
Line 28: | Line 26: | ||
*#Step2 : Strip | *#Step2 : Strip | ||
*#Step3 : 1st Rolony + RAB7A | *#Step3 : 1st Rolony + RAB7A | ||
*#Step4-0 : Strip | |||
*#Step4 : RAB7A only | *#Step4 : RAB7A only | ||
*#Step5-0 : Strip | *#Step5-0 : Strip | ||
Line 35: | Line 34: | ||
*#Step7-0 : Strip | *#Step7-0 : Strip | ||
*#Step7 : ACTB + RAB7A only | *#Step7 : ACTB + RAB7A only | ||
Line 44: | Line 42: | ||
=====Summary===== | |||
{| {{table}} border = 1 | |||
| | |||
|| | |||
| ACTB | |||
|| | |||
| (%) | |||
|| | |||
| RAB7A | |||
|| | |||
| (%) | |||
|- | |||
| w/i 1st Rolony | |||
|| | |||
| 74 [[Media:Exp2_DyeTest.lif_Step5_1stRolony_ACTB_20x_2k_Cy5_ATTO488_ch01_MIP.tif|Raw]] | |||
|| | |||
| 1.45 | |||
|| | |||
| 114 [[Media:Exp2_DyeTest.lif_Step3_1stRolony_RAB7A_20x_2k_Cy5_ATTO550_ch01_MIP.tif|Raw]] | |||
|| | |||
| 2.25 | |||
|- | |||
| 1 probe only | |||
|| | |||
| 157 [[Media:Exp2_DyeTest.lif_Step6_AddACTBOnly_20x_2k_Cy5_ATTO488_ch01_MIP.tif|Raw]] | |||
|| | |||
| 3.08 | |||
|| | |||
| 260 [[Media:Exp2_DyeTest.lif_Step4_AddRAB7AOnly_20x_2k_Cy5_ATTO550_ch01_MIP.tif|Raw]] | |||
|| | |||
| 5.13 | |||
|- | |||
| ACTB+RAB7A | |||
|| | |||
| 130 [[Media:Exp2_DyeTest.lif_Step7_AddACTB_RAB7A_20x_2k_ATTO488_550_ch00_MIP.tif|Raw]] | |||
|| | |||
| 2.55 | |||
|| | |||
| 268 [[Media:Exp2_DyeTest.lif_Step7_AddACTB_RAB7A_20x_2k_ATTO488_550_ch01_MIP.tif|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===== | |||
*Step1 : RAB7A only, | *Step1 : RAB7A only, | ||
**RAB7A, Count = 514, Overlapped = 160 | **RAB7A, Count = 514, Overlapped = 160 | ||
Line 69: | Line 128: | ||
**ACTB, Count = 234, Overlapped = 130 | **ACTB, Count = 234, Overlapped = 130 | ||
**RAB7A, Count = 463, Overlapped = 268 | **RAB7A, Count = 463, Overlapped = 268 | ||
=====Pictures===== | |||
{| {{table}} | |||
| 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|500px]] | |||
|| | |||
| [[File:Exp2_DyeTest.lif_Step5_1stRolony_ACTB_20x_2k_Cy5_ATTO488_ch01_MIP.tif_composite2.png|500px]] | |||
|} | |||
{| {{table}} | |||
| 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|500px]] | |||
|| | |||
| [[File:Exp2_DyeTest.lif_Step4_AddRAB7AOnly_20x_2k_Cy5_ATTO550_ch01_MIP.tif_composite2.png|500px]] | |||
|} | |||
{| {{table}} | |||
| 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|500px]] | |||
|| | |||
| [[File:Exp2_DyeTest.lif_Step7_AddACTB_RAB7A_20x_2k_ATTO488_550_ch01_MIP.tif_composite2.png|500px]] | |||
|} |
Latest revision as of 23:24, 7 November 2013
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 |