25/Nov/2011
probeの解離定数を求める。
I am gonna determine Kd value.
2 units/ml Bc-PLC
2/5 units/ml Bc-PLC
2/5*5 units/ml Bc-PLC
2/5*5*5 units/ml Bc-PLC
2/5*5*5*5 units/ml Bc-PLC
2/5*5*5*5*5 units/ml Bc-PLC
2/5*5*5*5*5*5 units/ml Bc-PLC
2/5*5*5*5*5*5*5 units/ml Bc-PLC
0 units/ml Bc-PLC
At the same time,
I am gonna determine real amount of DAG level by brigh and dyer method.
18/Nov/2011
SMの量におけるD609の影響をMDCK細胞で調べる。
Effect of D609 on SM level in MDCK cells
1)ctrl
2)SMase
3)3hour D609 incubation
4)6hour D609 incubation
5)20hour D609 incubation
mCherry-lysenin conc is 50microg/ml.
1:30 3hour
4:30 6hour
6:00 20hour
SMase 1 units/ml 10 min. After that, fixed.
What I have to prepare is that
HBSS and 3 % PFA
Effect of DGAT inhibitor on DAG level in MDCK cells.
1microM A922500 was used for this experiment.
2pm Add A922500 start incubation
4pm Add A922500 start incubation
5pm DAG probe incubation.
こんなかんじで
17/Nov/2011
Rat red blood cell experiment,
At first, I did the experiment as follows
1)1U/ml Pc-PLC + 200times annexin V + 9*108 cell/ml
result: hemolysis within 4 min
2)0.1U/ml Pc-PLC + 40 times annexin V + 9*108 cell/ml
result: hemolysis within 5 min
In this time, protocol was wrong.
3)real experiment
0.1U/ml Pc-PLC + 40 times annexin V + 9*108 cell/ml
I get annexin V fluorescence but it is not for living RBL cells.
At first, RBC was subjected to hemolysis, become ghost, and then become fluorescence.
4)0.1U/ml Bc-PLC + 40 times annexin V + 9*108 cell/ml
RBC rarely give hemolysis and no fluorescence.
And I tryed the experiment whether Bc-PLC makes DAG on the plasma membrane in RBC in a Bc-PLC conc. dependent manner.
1) 800 microl RBC (9*108 cell/ml)
2) Add DAG probe 24 microl here
And
3)2microl 100 units/ml Bc-PLC was added to the 8 microl HBSS.
4)2microl of this solution was added to next tube, which has 8 microl HBSS.
5)this step is done one after another.
this means that 5 time solution is gonna prepare.
Next
in each tube, 75 microl RBC solution was added and is incubated for 10 min @r.t.
after that, fixed 6% (final 3%), and incubated for 15min. and spin down.
こんな感じでやりましたよーん。
30/Oct/2011
D609の濃度
50microg/ml
Offered as 5 mg (sc-201403) and 25 mg (sc-201403A) sizes
Synonym: O-Tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt
CAS Number: 83373-60-8
Molecular Weight: 266.5
Molecular Formula: C11H15OS2K
Purity: 98%
Physical Appearance: Off-white solid
Solubility: Soluble in water (25 mg/ml)
I used D609 with the concentration of 188microM
以下のものをやってみる。
Other experiments
1. living MDCK cells with alpha PMA and PMA
2. DAG probe calibration with Bc-PLC
Bc-PLC 20 units/ml
Bc-PLC 10 units/ml
Bc-PLC 5units/ml
Bc-PLC 0units/ml
D609
3. fixed MDCK cells with SMase
noSMase
SMase
SMase+D609
SMase+BcPLC
4. living MDCK cells with SMase + Bc-PLC
5. SM amount with SMase or nojirimycin
6. DAG amount with SMase or nojirimycin
111016:もう一度最後の実験
以下の実験を２回繰り返す。
それぞれについて１０個の細胞をとる。
それでanalyzeして終わり。
Thinking about biological significance, I am gonna try this experiment as follows.
1. noSMase
2. SMase
3. SMase+D609
4. SMase+BcPLC
16/June/2011
Now I started to write the papers.
But I have several experiments I have to do and I want to do.
1. test if the DAG which exist already on the membrane is abolished by incubated with D609.
2. I am gonna take beautiful data of cholesterol, DMS and NB-DNJ.
SMS2がPC-PLCであるか否か？とういうことに関係するのですが、
D609処理した細胞で、ライセニンで染まるかどうかという実験を過去にラボでやった人はいらっしゃいますか？
あと、SMS1およびSMS2が欠損したMEF-ZS2とSMS1もしくは、SMS2が発現させた細胞では、ライセニンで染めるとどのように違いがでますか？
こちらについては、やっているのではないかと思い聴いて見ました。
And then I want to get the PC-PLC antibody.
20May2011
I am gonna do the experiment.
I don't miss to write but in the previous experiment.
UV was irradiated to MDCK cells for 1 min.
30 min, 1hour 2hout later, the cells were fixed and observed.
30min there is 30% cells in which DAG probe was translocated.
But not so obvious. So I am gonna do 10 min incubation after UVB irradiation.
Because some papers says that 10 min is more DAG.
So,
1. UVB 10min
2. UVB D609 10 min
3. No UVB
And then
the previous experiment that when MDCK was fixed, DAG probe localized at the plasma membrane incubated with 12.5 Bc-PLC 10 min.
So, I need to play around the concentration of Bc-PLC.
4. Bc-PLC 0 microunit/ml
5. Bc-PLC 0.1 microunit/ml
6. Bc-PLC 0.5 microunit/ml
7. Bc-PLC 1 microunit/ml
8. Bc-PLC 5 microunit/ml
9. Bc-PLC 12.5 microunit/ml
And then,
U73122 doesn't abolish ATP-induced DAG on the outer leaflet of plasma membrane. So, I suspect PAP-induced DAG. I am gonna use the propranolol.
10. ctrl
11. D609
12. D609+ATP
13. D609+ATP+propranolol
At the same time,
I am gonna use the MDCK cells expressing mGluR5 and bath application of glutamate.
14. ctrl
15. glutamate
06May2011
I gonna do the experiment about DAG flip in fixed cells.
A. EYFP-C1AB and DsRed
B. EYFP-C1AB and DsRed with 10mM MbCD + 12.5unit/ml BcPLC
C. EYFP-C1AB and DsRed with 20microM Fumonisin B1 + 12.5unit/ml BcPLC
D. EYFP-C1AB and DsRed with 50nM ISP1 + 12.5unit/ml BcPLC
E. EYFP-C1AB and DsRed with 6 microM DMS + 12.5unit/ml BcPLC
F. EYFP-C1AB and DsRed with 100 microM NB-DNJ + 12.5unit/ml BcPLC
G. EYFP-C1AB and DsRed with Bc-PLC as ctrl
H. EYFP-C1AB and DsRed with PMA
28Apr2011
How to irradiate UVB (290-320nm)
1. reduce the volume of medium, DMEM 100 microl.
Go to program
2. Set 10 mJoules/cm2 for 1min
3. replace the medium with appropriate media.
That's it.
27Apr2011
Now Francoise kindly decide the condition of UVB irradiation.
So I am doing the following experiments under the condition.
I will observe DG probe and PKCdelta antibody accumulation.
1. Ctrl(negative) Just MDCK cells.
2. Ctrl(positive) PMA
3. UBV and leave 30 min
4. UBV and leave 1 hour
5. UBV and leave 3 hour
7. UBV and leave 30 min with D609 (D609 nothing after UVB)
8. UBV and leave 1 hour with D609 (D609 nothing after UVB)
9. UBV and leave 3 hour with D609 (D609 nothing after UVB)
10. UBV and leave 3 hour with D609 (D609 still incubation after UVB)
11. no D609
12. D609+100microM ATP
13. D609+100microM ATP+1microM U73122
22Apr2011
Thinking about the experiment I have to do.
1. I can get PKCgamma antibody within next week.
So I gonna do the following experiment.
A. EYFP-C1AB, PKCgamma antibody, DsRed.
B. EYFP-C1AB, PKCgamma antibody, DsRed with SMase.
C. EYFP-C1AB, PKCgamma antibody, DsRed with SMase and D609.
2. check whether DAG flop is inhbited in the presence of U73122.
Actually when I did this expeirment previously using 100 microM U73122,
unexpectedly more bright fluorescence comes up.
Maybe some artifact.
So I gonna reduce the concentration of U73122.
A. 100 microM ATP
B. 100 microM ATP, 10 microM U73122
C. 10 microM ATP
D. 10 microM ATP, 10 microM U73122
3. I am gonna observe the effect of MbCD, FumonisinB1, ISP1, DMS and NB-DNJ in fixed cells.
A. EYFP-C1AB and DsRed
B. EYFP-C1AB and DsRed with 10mM MbCD + 12.5unit/ml BcPLC
C. EYFP-C1AB and DsRed with 10microM(?)Fumonisin B1 + 12.5unit/ml BcPLC
D. EYFP-C1AB and DsRed with 50nM ISP1 + 12.5unit/ml BcPLC
E. EYFP-C1AB and DsRed with 6 microM DMS + 12.5unit/ml BcPLC
F. EYFP-C1AB and DsRed with 100 microM NB-DNJ + 12.5unit/ml BcPLC
07Apr2011
Thinking about discussion with Francoise.
1. I am giving Francoise MDCK cells.
2. I gonna show the paper I am preparing now.
3. I gonna give her D609, rottelin Reiko has.
4. I gonna give her the paper relationship with D609 and apoptosis, with rottelin and apoptosis, and MDCK and apoptosis, SMase and apoptosis.
25/Mar/2011
Thinking about biological significance, I am gonna try this experiment as follows.
1. noSMase
2. noSMase+U73122
3. noSMase+D609
4. SMase
5. SMase+U73122
6. SMase+D609
7. SMase+BcPLC
8. SMase+BcPLC+U73122
Express EYFP-C1AB, DsRed and PLCepsilon-HA
Express EYFP-C1AB, DsRed and PLCepsilon-HA(dominega)
Express EYFP-C1AB, DsRed and PLCalpha-HA
Express EYFP-C1AB, DsRed and PLCalpha-HA(dominega)
24/Mar/2011
And then, I found another contraversy.
In IPS1 experiment, flip doesn't occure even though without SM.
I need to do same experiment with SMase.
22/Mar/2011
I am gonna do the following experiments from the data I took on 15/Mar/2011.
At first, 10 microM U73122 experiment doesn't work.
So I am gonna do increase its concentration or decreases in ATP concentration.
This experiment is very critical to say that the EYFP-C1AB fluorescence is caused by DAG drived from PI-PLC.
1. 100 microM ATP
2. 100 microM ATP, 100 microM U73122
3. 100 microM ATP, 100 microM U73343
4. 10 microM ATP
5. 10 microM ATP, 100 microM U73122
6. 10 microM ATP, 100 microM U73343
Another point I have to mention is that 24 hour incubation with U73122 did not give the difference in outer DAG signal compared to ctrl.
This means that PI-PLC doesn't contribute to outer DAG.
But, I can see PI-PLC dependent outer DAG sigal.
So I want to make clear the signal from PI-PLC caused by ATP addition is transient or sustained. I guess this signal is transient.
I am gonna do 10min, 30min, 1 hour, 2 hour. In some case I am gonna remove the ATP in the middle of reaction.
15/Mar/2011
I am gonna take reproduciblity.
1. MDCK Cameleon with ATP no D609
2. MDCK Cameleon with ATP with D609
3. MDCK control no D609
4. MDCK D609 only
5. MDCK D609 ATP
6. MDCK D609 ATP U73122
7. MDCK D609 ATP U73343
8. MDCK D609 ATP R59949
9. MDCK D609 ATP SMase
10. MDCK D609 PMA
11. MDCK D609 DiC8
12. MDCK C1AB inner no D609
13. MDCK C1AB inner no D609 ATP
14. MDCK C1AB inner D609
15. MDCK C1AB inner D609 ATP
16. MDCK mGluR no D609
17. MDCK mGluR D609
18. MDCK mGluR D609
Result: Patially success.
I got good result about that in the presence of D609 and ATP the DAG signal was detected, but no signal in the presence of D609 only. Of course, no D609 gives very bright fluorescence. But disappointed point is that U73112 did not inhibit the fluorescence which is caused by addition of ATP. Interpretation of it is due to week effect of U73122. I need to increase in concentration of U73122 or decrease of concentration of ATP.
And then, the points I need to condider is that in the presence of U73122, which is 24 hours incubation, DAG signal did not decreases. But transient increase caused by inner DAG caused by ATP through PI-PLC is detected. How do I come to term with these.
09/Mar/2011
The idea to observe DAG flop.
1. no D609
2. ctrl
3. ATP 10 min
4. ATP U73122 10 min
5. ATP R59949 10 min
6. ATP SMase 10 min
7. PMA 10 min
8. DiC8 10 min
9. Inner C1AB ATP
10.Inner C1AB ATP
1. About 9 and 10, C1AB is transfected one day before.
2. 2-10 is incubated with D609 for 6 hours.
3. 1067microl(97times11)HBSS is prepared
4. 33microl C1AB is put, means total 1100microl
5. 100microl pick up. Number1
6. 4microl D609 is put, total 1004 microl
7. Divide in half
8. first half, 5 microl ATP is added.
9. 100 microl pick up. Number2.
10. From remaining 400microl, each 100 microl is taken, add 1 microl PMA(10microM) and 1 microl DiC8 (100microM).Number 7 and Number 8.
11. About second half, 4microl ATP is added.
12. 100microl is picked up, Number3.
13. 1 microl U723122(15microM), R59949(10microM) or 2microl SMase(1unit) is added to each 100 microl, Number 4,Number5 and Number 6.
Result:In some part it is works well.
But I missed to enter D609 in the cell which was supposed to be in the presence of U73122. So I need to do again about U73122.
And My concern is C1AB probe expressed into the cells localized to the membrane in the presence of only D609.
I need to chech this is really real or not.
And I would be better to confirm that P2Y2R stay on the surface on the plasma membrane, by doing measure increase in calcium concentration.
c
04/Mar/2011
I prepared WR19L cells, lymphocyte.
I am gonna do the following experiments.
I am gonna add 10 microM PMA, 100 microM DiC8, 12.5 units/ml Bc-PLC, 10 microM C6-Cer mixed with EYFP-C1AB probe.
Experimental procedure
WR19Lcells were spind down and collected.
Cells were in total 815 microl(163microl*5 in each)HBSS solution.
Add 25microl 3.8mg/ml EYFP-C1AB.
2microl 1mM PMA, 1mM alphaPMA, 100mM DiC8, 8.5microl of 12.5unit/ml Bc-PLC
are put in the 1.5 ml tubes in advace.
168microl solution containing EYFP-C1AB is added in each tube and incubate 10 min.
30micro 20% PFA is added and fix for 10 min.
After that, spine down cells and remove supernatant.
Add 50mM NH4Cl for 5min.
Spine down and suck up supernatant.
Add 100microl HBSS.
Result:I could do it! I can see brighter fluorescence in PMA than that in control.About DiC8, It seems that there is no difference compared to control.About Bc-PLC, I could see very bright fluorescence in the cells which is aggregated, indicating that DAG is important for membrane fusion.
04/Mar/2011
I prepared WR19L cells, lymphocyte.
I am gonna do the following experiments.
I am gonna add 10 microM PMA, 100 microM DiC8, 12.5 units/ml Bc-PLC, 10 microM C6-Cer mixed with EYFP-C1AB probe.
Experimental procedure
WR19Lcells were spind down and collected.
Cells were in total 815 microl(163microl*5 in each)HBSS solution.
Add 25microl 3.8mg/ml EYFP-C1AB.
2microl 1mM PMA, 1mM alphaPMA, 100mM DiC8, 8.5microl of 12.5unit/ml Bc-PLC
are put in the 1.5 ml tubes in advace.
168microl solution containing EYFP-C1AB is added in each tube and incubate 10 min.
30micro 20% PFA is added and fix for 10 min.
After that, spine down cells and remove supernatant.
Add 50mM NH4Cl for 5min.
Spine down and suck up supernatant.
Add 100microl HBSS.
18/Feb/2011
I am gonna follow Malhotra's procedure.
I prepared the liposomes as follows.
1. POPC only=60
2. POPC/PS=60/20
3. POPC/GM1=60/20
These liposome includes 2mol% Rhodamine-PE
1. POPC(0.2micromol) avanti 13.7mM 14.6microl
2. PS(0.04microl)avanti 100721 2.3mM 0.45 microl
3. GM1(0.04micromol) sigma G7641 1mM 1.128 microl
4. Phodamin-PE(4nmol) Avanti 0.15 mM 28 microl
1. I diluted these liposomes in 666microl, 888microl, 888microl in each.
This means that 300 microM liposomes.
2. I am gonna pick up 100 microl in each.
3. EYFPC1AB domain is 1 mg/ml
I am gonna add 1microl into 100 microl.
This means that 10 microg/ml.
4. About DiC8, I have 1M DiC8 now (DMSO).
I am gonna dilute this solution 200 times(PBS(-)), this means 5mM.
Add 1microl into 100 microl.
I want to prepare as follow.
A. POPC
B. POPC+probe
C. POPC+probe+DiC8
D. POPC/PS
E. POPC/PS+probe
F. POPC/PS+probe+DiC8
G. POPC/GM1
H. POPC/GM1+probe
I. POPC/GM1+probe+DiC8
5. FRET mesurement.30 min incubation.
excitation 488 nm emission 500-650nm
6. Flotation assay (I can only use 4 samples for ultracentrifugation)
B. POPC+probe
C. POPC+probe+DiC8
H. POPC/GM1+probe
I. POPC/GM1+probe+DiC8
7. Normalize with fluorescence of liposomes.
8. Apply sample solution to SDS-Page.
9. Stain with GFP antiboly.
06/Feb/2011
I couldn't make liposomes previously in 03/Feb/2011.
However, I leave these lipid films for 1 week in the room condition.
This might cause some bad effect on these membranes.
So, I gonna make liposome again with same condition with littele modification.
Because previous study use fluorescence PE was used 2mol%.
I am gonna increase the volume of Rhodamine-PE from 0.5 to 2 mol%.
So,
1. POPC only
2. POPC/DAG=60/6
3. POPC/DAG/GM1=60/6/20
4. POPC/DAG/PS=60/6/20
5. POPC/PS=60/20
6. POPC/GM1=60/20
Phodamine-PE is inluded all 2 mol%.
1. POPC(0.2micromol) avanti 13.7mM 14.6microl
2. PODG(0.033micromol) avanti 800815C 3.4 mM 9.8 microl
3. PS(0.04microl)avanti 100721 2.3mM 0.45 microl
4. GM1(0.04micromol) sigma G7641 1mM 1.128 microl
5. Phodamin-PE(4nmol) Avanti 0.15 mM 28 microl
So then, I am gonna prepare following.
1. Take 102 microl POPC and 196 microl PE.
2. Mix and take 42.6 microl each in 6 tubes.
3. 9.8microl DAG is added 2,3,4 tubes.
4. 1.1microl GM1 is added 3,6 tubes.
5. 0.5microl PS is added 4,5 tubes.
result: I try sonication extensivly. But didn't make liposomes containing DAG
even though biophysical journal paper shows that they could make liposomes.
03/Feb/2011
Lipid filmes prepared in 20/Jan/2011 was voltexed.
But 5,6 didn't make liposomes.
2,3,4 did but rim of glass tube have fluorescence lipids, I am afraid of phase separation of DAG on the glass tube.
Only 1 made liposomes well.
20/Jan/2011
I did the experiment of 09/Jan/2011
But this experiment didn't work well.
Even positive control didn't work well, which is same result of ctrl.
Toshi-sensei said that the distance between fluorescent PE and EYFP-C1AB doesn't become close enough.
So I am gonna give up this experiment.
I am gonna do following expeiment.
I will prepare following liposomes.
1. POPC only
2. POPC/DAG=60/6
3. POPC/DAG/GM1=60/6/20
4. POPC/DAG/PS=60/6/20
5. POPC/PS=60/20
6. POPC/GM1=60/20
Phodamine-PE is inluded all 0.5 mol%.
1. POPC(0.2micromol) avanti 13.7mM 14.6microl
2. PODG(0.033micromol) avanti 800815C 3.4 mM 9.8 microl
3. PS(0.04microl)avanti 100721 2.3mM 0.45 microl
4. GM1(0.04micromol) sigma G7641 1mM 1.128 microl
5. Phodamin-PE(1nmol) Avanti 0.15 mM 7 microl
So then, I am gonna prepare following.
1. Take 102 microl POPC and 49 microl PE.
2. Mix and take 21.6 microl each in 6 tubes.
3. 9.8microl DAG is added 2,3,4 tubes.
4. 1.1microl GM1 is added 3,6 tubes.
5. 0.5microl PS is added 4,5 tubes.
09/Jan/2011
I read a paper in which they made liposomes. I will modify a little bit.
And I am gonna detect FRET signal. ex: 488 nm, em 560 nm.
1.POPC/BODIPYPE(530/560)=60/0.6
2.POPC only (as ctrl)
3.POPC/PS/BODIPYPE(530/560)=60/6/0.6 (positive ctrl)
4.POPC/GM1/BODIPYPE(530/560)=60/6/0.6
5.POPC/GM1=60/6 (as ctrl)
Each is divided into two samples. One is for ctrl. The other is for Bc-PLC addition.
1. POPC(0.2micromol) avanti 13.7mM 14.6microl
2. BODIPYPE(530/560)(0.004micromol) Molecular probe 1 mM 2microl
3. PS(0.02microl)avanti 100721 2.3mM 0.245 microl
4. GM1(0.02micromol) sigma G7641 1mM 0.564 microl
03/Jan/2011
I did the condition of 02/Dec.
But this lipid mixture did not make liposomes more than previous condition.
So Next thinking is that remove cholesterol.
That menas that POPC/SM/DAG=60:20:10 and so on.
And I did the experiment in which I see the effect of C1AB on cell growth.
MDCK and HEK cells.
I cultured HEK cells as following condition.
cell concentration is 1.62*106 cells/ml. 10microl solution was added to 500microl medium. After that 10microl 3.8mg/ml C1AB was added.
Ctrl is only 10microl 200mM imidazole buffer solution.
About HEK cells, 3.0*105 cells/ml was prepared. 30microl this solution was added to 500microl DMEM solution. After that, 25microl 3.8mg/ml C1AB solution was added.
1. POPC(0.2micromol) avanti 13.7mM 14.6microl
2. brainSM(0.066micromol) avanti 860062C 31.4mM 2.1 microl
3. chol(0.2micromol) unknown 10.2mM 19.6microl
4. PODG(0.033, 0.066micromol) avanti 800815C 3.4 mM 9.8, 19.6 microl
So, I made such liposomes.
1. POPC=60
2. POPC/DAG=60/5
3. POPC/DAG=60/10
4. POPC/SM=60/20
5. POPC/SM/DAG=60/20/10
6. POPC/SM/DAG=60/20/20
02/Dec/2010
It turns out that these lipid mixture I prepared in 30/Dec did not form liposomes.
So, I asked Toshi-sensei how to make liposomes well.
うえだくん、
コレステロールを増やせばうまくいくかも知れません。
POPC/SM/chol/DAG=60:20:60:10とか。
試してみてもらえますか？
小林
So I made liposome as follows.
1. POPC(0.2micromol) avanti 13.7mM 14.6microl
2. brainSM(0.066micromol) avanti 860062C 31.4mM 2.1 microl
3. chol(0.2micromol) unknown 10.2mM 19.6microl
4. PODG(0.033, 0.066micromol) avanti 800815C 3.4 mM 9.8, 19.6 microl
So, I made such liposomes.
1. POPC/SM/chol=60/20/60
2. POPC/SM/chol/DAG=60/20/60/10
3. POPC/SM/chol/DAG=60/20/60/20
30/Dec/2010
I prepared liposomes newly.
SM was included because SM is important for distribution of DAG on the membrane.
And then, I mimicked the outer leaflet of the plasma membrane.
So,
I used as follows
1. POPC(0.6micromol) avanti 13.7mM 43.8microl
2. brainSM(0.2micromol) avanti 860062C 31.4mM 6.3 microl
3. chol(0.2micromol) unknown 10.2mM 19.6microl
4. PODG(0.1, 0.2, 0.3,0.4 micromol) avanti 800815C 3.4 mM 29.4, 58.8,88.2,117microl
So, I made such liposomes.
1. POPC/SM/chol=60/20/20
2. POPC/SM/chol/DAG=60/20/20/10
3. POPC/SM/chol/DAG=60/20/20/20
4. POPC/SM/chol/DAG=60/20/20/30
5. POPC/SM/chol/DAG=60/20/20/40
28/Dec/2010
EYFP-C1AB 1.77 mg/ml now (M.W=38,000)
I took 3 microl
this means 1.77 mg*3microl/1000microl=5.31 microg.
So 5.31/38000=0.0001397micromol=0.1397nmol.
1 cell have 1 fmol DAG.
glass base dish maybe has 10000 cells/1 well.
So 1 fmol*10000=10pmol DAG.
but outer DAG is less assume 1%.
So 100fmol DAG.
liposome
19mM DOPC 52.6microl
this means 19 mmol/1000000microl*52.6microl=1micromol.
3.2mM DODG 12.5microl
this means 3.2mmol/1000000microl*12.5microl=40nmol.
These two are mixed, so 4%mol DAG.
suspended in 200microl HBSS.
100microl picked up, means 500nmol PC 20 nmol DAG.
vesicle contains two side, so 250nmol PC 10nmol DAG
I did liposome experiment, but result is mysterious.
In the presence of DAG in PC liposome, fluorescence light is brighter than that in the presence of only PC liposome.
I think about this.
Basically, I don't know what happen, but there is some hint.
1. cells were ripped off when both liposome was added, this means that PC works as detergent because I add a lot of liposome, 500 nmPC.
So I need to reduce the amount of PC liposome.
2. In this experiment, I add 4mol% DAG in PC liposome. But other experiment, for example SM-lysenin, give 20mol% DM into PC liposome.
So I can add more DAG, maybe at least 10%, possbly 20%-40%. As you know,
If DAG included more, this membrane doesn't make a liposome. So I need to take care about it.
Next time, I am goning to prepare the liposome,
リポソームですが、1mMを1ml位用意して、
蛋白の濃度にもよると思いますが、
こちらの実験では、ＧＦＰ－Ｌｙｓの場合、
いつも、５０倍希釈で染色しているので、
(１マイクロ/５０マイクロＰＢＳ）
１マイクロに、５０マイクロのリポソームを混ぜて、
３７度で３０分してから、それで染色していました。
１ｍＭはかなり濃い濃度なので、タンパク質はすべて
結合すると考えています。
さっき探していた論文ですが、(準備中）
For the preabsorption experiment, Dronpa-θ-D4 or Dronpa-NT-Lys was incubated with MLVs at 37°C for 30 min prior to apply them to HeLa cells.
としか書いてありませんでした。
これで良いですか？
牧野
Reply Forward
Reply |Ueda Yoshibumi to Asami
show details Dec 17 (7 days ago)
リポソームを作るとき、はじめに、クロロホルムに溶けた脂質を遠沈管の中に加えて、
窒素で飛ばしてからPBSなどを加えてベシクルにするんだよね？
だいたい、クロロホルムに溶けた1mM脂質（例えばDOPC)を何μl位遠沈管に入れて乾かしていますか？
それは脂質のストック濃度によると思います。
１０ｍＭの脂質ストックなら１００ulだし、２０ｍＭなら５０です。
それで乾いてから1mlのＰＢＳをいれて、
最終濃度1mMリポソームが 1mlできるということです。
Reply Forward
Reply |Ueda Yoshibumi to Asami
show details Dec 17 (7 days ago)
１ｍMだったら脂質を１ｍｌ入れているっていうことだね。
かなり多い量を加えているんだね！
なるほど、わかりました。
Reply |Asami Makino to Ueda
show details Dec 17 (7 days ago)
でも、結局リポソームは５０マイクロＬとか使わないから、
1mlも作る必要ないと思う。
２００マイクロでも作れば充分なんじゃないかな。
ただ、こちらにある脂質のストックはほとんど10mM以上あるので
たくさんある脂質は少量はかり取るのが大変だから、
たくさん使ってもいいと思います。
From: Ueda Yoshibumi
To: Asami Makino
Sent: Friday, December 17, 2010 3:31 PM
Subject: Re: MLV
なるほど、了解しました！
So, I prepared vesicles.
I use 10microg/ml YFP-C1AB to stain the cells.
YFP-C1AB M.W. 38kDa=38,000
This means 0.000263micromol/ml
I add 100microl, So total amount is 0.02nmol.
I want to include DAG into the vescle
And then, 1 cells has 1 fmol DAG. glass based dishes is maybe 10 pmol DAG.
最終更新：2011年11月25日 13:31
