I have problem on Actin band - SDS-PAGE and Western blot (May/12/2006 )
Hi all... i'm newest for SDS-PAGE
I have prepare human fibroblas cell line and I'm finding Actin 43kDa and hMTH1 18kDa but I'm not sure where is my protocol have any mistaked or not.. 
After I remove cells I put on ice and then I mixed my samples with laemmli buffer which was prepared fresh and kept in refregerator 4 C, after that isonicated sample-leammli 30 sec then I keep sample-leammli buffer at -20 C for running PAGE next time. 10^5 cells for 20 ul leammli buffer
My leammli Buffer(for prepare 4 ml)
H2O...................1.75 ml
0.5 M Tris-HCl pH6.8........0.5 ml (store 4 C)
Glycerol..........0.4 ml
10% SDS..........0.8ml (RT)
2-Mercaptothanol..........o.2ml
1% Bromphenolblue.............0.2ml (RT)
50mM PMSF...........160 ul (-20 C)
0.5 M EDTA..............2 ul (4 C)
Protease inhibitor 25X .............0.4 ml (4 C)
Q : what will happen with cells/protein when I mixing leammli buffer with sample?? or what key word could I find about this infomation. that I know SDS will break bond and membrane of cell but I done know in deep deteils ...
Q: what's importance/function of protease inhibitor I have read it but I don't understand - -*
I have boiled sample-leammli 1 minute and put on ice before load into gel and I loaded samples 20 ul each well
I use 15%separate gel and 5 % stacking gel. I have degased about 15 min at least..
I run gel at 100 V 1 hr. then I transfer protein to PVDF membrane 150mA 30V over night on ice.
Q 1: How can I design to use voltage or it depends the molecular weight of protein or concentration of gel or anything else?? because I read paper of somebody use in many of voltage or mA. what's the reason ?
I have used TBST for washing and blocking with 5% milk in TBST 1.30 hr
after that I use rabbit anti MTH1 polyclonal 1:1000 (Novus biological) goat anti actin polyclonal 7:10000(santacruz) for 1 ab with 3% milk - TBST in both of them
donkey anti rabbit HRP and anti goat HRP (santacruz) for 2 ab 1:2000 with 3% milk - TBST
for the actin band sometimes disappear but sometimes clearly.
for the hMTh1 band I can see band at 18 kDa and 13 kDa
Q 2: in the 13 kDa ,is it another protein or hMth1 degradation?? why does it degradation ??
now i'm repeating my protocol ... maybe i can compare with old one
although,hopefully somebody give me advice, thank in advance 
and sorry if my explaination it's not clearly bcoz my English quite weakly : P
Q : what will happen with cells/protein when I mixing leammli buffer with sample?? or what key word could I find about this infomation. that I know SDS will break bond and membrane of cell but I done know in deep deteils ...
SDS an b-Mercaptoethanol denature protein
Glycerol increases density so the sample sinks into the bags of the gel
Bromphenolblue is a dye so you see the samples running through the gel
Q: what's importance/function of protease inhibitor I have read it but I don't understand - -*[/b]
Proteases are inhibited so your proteins don`t get degraded...
I have used TBST for washing and blocking with 5% milk in TBST 1.30 hr
after that I use rabbit anti MTH1 polyclonal 1:1000 (Novus biological) goat anti actin polyclonal 7:10000(santacruz) for 1 ab with 3% milk - TBST in both of them
donkey anti rabbit HRP and anti goat HRP (santacruz) for 2 ab 1:2000 with 3% milk - TBST
for the actin band sometimes disappear but sometimes clearly.
for the hMTh1 band I can see band at 18 kDa and 13 kDa
You loaded the same volume of sample in each well but didn't measure the protein concentration?!? To compare the amount of your protein (hMth1) you must load the same amount of whole protein.
Actin is the loading control to make sure there was no pipetting error when loading the gel:
If you load the same amount of whole protein in each well (not the same volume!) the signal should be of the same intensity for each lane.
If your samples are already in lämmli I think you can't use bradford or bca assay but tcato measure the protein concentration of your samples.
Q 2: in the 13 kDa ,is it another protein or hMth1 degradation?? why does it degradation ??
Are you sure your first antibody (actin or hMth1) isn`t just unselective?
Though it is strange that the 13kD signal is stronger when the 18kD signal is weaker.
Shouldn't be degradation though cause your buffer contains protease inhibitors....
[/quote]
[quote name='Sawaddee' date='May 15 2006, 01:46 PM' post='51817']
[quote name='kylvalda' post='51779' date='May 15 2006, 10:54 AM']
Hello Kylvalda
Thank you very much all your advice they are really helpful to me 
For my samples, I've calculated amount of cell. In each samples, there are amount of cells by average 3x10^5 cells then I combined with leammli buffer 60 ul then I kept them at -20.
New Q : How long could I keep sample+leammli or should I prepare fresh samples?? because I have kept them about 2 month ago before I run Gel electrophoresis.
If you keep them at -20° it should be no problem but a lot of ´thawing and refreezing isn't good for the samples so if you have a large volume it'd be better to aliquot the samples into smaller volumes...
When I loaded samples, I use the 50 ul syringe and loaded samples 20 ul in each well.
I still think you should mesure the protein concentration and load an equal of total protein not equal volume...
From this picture
right side : I use only 1 ab and 2 ab for hMth1
there are my control samples but different time when they were prepared(I have to say by honesty am not sure what I did any mistake when I prepare this cell.) you will see well no.3(left to right) it was irradiated 1Gy and incubated time 5 hours before I mixed with leammli but no.1 and 2 it's my control I think they have something wrong.
left side : I use only 1ab and 2 ab for Actin
[/quote]
[i] are you sure? Actin is about 42kD and should then be on the right side of this picture and your hMth1 ón the left.
What do you mean by "irradiated 1Gy"? Because Lane 3 looks best to me (just a Signal at 18kD and none at 13kD)...
but what is tcato this assay I don't know if it is one kind of measure to protein concentratio I will look it up..but i haven't sought yet
Sorry for misstyping it means tca (stands for trichlor acetic acid)
For protocols on bradford, bca or tca search here:
Protocol online
for me I run gel 100 V 2 hours, and run western blot transferm I was setting 150 mA 30 V overnight.
I really don't know...
Well, with the gel you just have to make sure that your samples run long enough to separate in the gel and don't run through (prestained marker is a great help here)
And for the blotting: if you run it too long your proteins pass the membran and are lost in the buffer, another problem is that the blot shall not run hot.
Me, I blot for 1,5 hours 350 mA
In your case (blotting overnight) I strongly suggest to use a second membrane next time which you just lay over your regular membrane to check if proteins are blotting through...