whats the problem of my western blot? - (Jun/02/2005 )

Hi
i have several problem encountered in the western blot.

1.look at this image,
the first lane is protein marker, the secondary and third lanes are the same samples but different treatment before loading. for the secondary land, i did not add reducing agent and did not boil; while in the third lane, i added beta-mercap as reducing agent and boiled for 5min.

actually the aim of my target bands are two bands, one is about 65KD, which is the proenzyme of heparanase, the other is around 50kD, the active form of heparanase. but now from my result, i could detect strong band around 30. the antibody i used is monocolonal antiheparanase.

does any one has any suggestion to use what kind of treatment condition for the next run of experiment? do i need just boil the sample but neglect the reducing agent?

2. for the boil of the sample, i want to ask wheather i need to boil the sample just after extraction(extraction buffer: RIPA, with 0.1%SDS) or i need boil the sample after extraction as well as after the adding of the loading buffer(with 4%SDS)?

i ask this question is because i always could not totally lysis the cell. when i add RIPA in the cell pellet(5X10 power 6/ml), i always see some sticky gel -like pellet? is it the not efficiency of the lysis buffer or the problem of DNA? will there be the sticky matter that cause the sample protein concertration is only around 1mg/ml?undefined

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hi
i always boil my samples in the B mercapto loading buffer. Hence i ould suggest it's better for you to boil in your loading buffer. If you encounter problems in cell lysis, it's probably because you don't use enough lysis buffer regarding quantity of cells. I would double the quantity of lysis buffer and see.
On the other side, my proteins concentrations are 4 to 10 µG/µl. so i don't think protein concentration is a problem...

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hi,
Thank you for your help.
This time iI added the 1ml of lysis buffer in 4X10^6 cells. the protein concentration of my sapmle only 1mg/ml, that is only 1/4 to 1/10 of your sample. because the well size, Icould only add 20µl sample and 20µl treatment buffer in each well. so the total protein in each lane is around 20µg. I am afraid my target protein is too less to be detected. will there be the problem?

for the amount of lysis buffer, orginally I used a lot, then after the test of concentration, it seams even lower, so usually at time I will concentrate the sample by vacumn. the problem cause is the salt concentration is too high in the sample, the gel will not run evenly, there will be smiles and the marker will also be squeezed like this figure i attached here.


so now in case the salt problem, I try not to use so many lysis buffer. I am not realized this can cause another problem.

by the way, do you have any suggestion of how many lysis buffer for how many cells? or how many lysis buffer in 35cm^2 petri dish?

recently I am performing the explant culture of E15 dosal root ganglion, after two days culture, i will also need collect the sample for the western blot. I also go the problem that the sample did not totally dissove for less lysis buffer or too high salt concentration after the concentration of the sample. I thought about the using centricon to desalt, but I am worry about the recovery of the protein.

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hi
when i see the coomassie staining, i would say you don't have many proteins. I run usually 20 to 25µg and i have more intensity. But maybe it's a problem of concentration.
lysis buffer depends on the quantity of cells. I use Approx 75 µl of lysis buffer for 3.5 petri dish.
For desalting your proteins and maybe get rid of the lysis buffer quantity, you can maybe make a TCA/Acetone precipitation after your extraction?

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