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[Mounir]

Hello all,
I'm a master's student in a Biology Departmant. I'm facing a trouble in pre-processing blood samples before using them in Western Blot technique. My aim is to examin the expression of Cathpsin-K, i.e. to measure (approx.) the serum level of this enzyme.
So if anybody can provide me with tips, I would be so pleased.
Best Regards.

[admin]

Originally Posted by Mounir

Hello all,
I'm a master's student in a Biology Departmant. I'm facing a trouble in pre-processing blood samples before using them in Western Blot technique. My aim is to examin the expression of Cathpsin-K, i.e. to measure (approx.) the serum level of this enzyme.
So if anybody can provide me with tips, I would be so pleased.
Best Regards.

Dear Mounir,
Welcome to the forum! you have posted in a place that few will be able to help, but I can move your post somewhere else and also help you here.

I assume Cathpsin-K is in the serum? not the blood cells in that case...

As you are using Blood, you would need to:

1) centrifuge the samples (you would need a large amount to begin with)

2) isolate the serum by taking only the supernatant (top layer)

3) it would be difficult to blot so you could load a large amount (or precipitate a larger amount of serum into a smaller volume and load that)

4) blot for that protein

is this a good guide or did I miss anything?

[Mounir]

Dear admin.,
First of all I would like to tell you that I'm pleased about your care. The steps you mentioned are themselves the ones I proceeded with. I was centrifuging the blood sample, isolating the serum, and then loading (5) or (10) microliters of the serum after mixing it with loading buffer. The former amount yeilded better bands in the gel but these are still so jammed in which you can't make a reading. I guess if we can get rid of certain kinds of proteins which are known to be found in the serum in high amounts, this may be a solution!!

Please advice,
Regards.

[Mounir]

Dear admin.,
First of all, I would like to tell you that I'm pleased about your care. The steps you mentioned are themselves the ones I proceeded with. I was centrifuging the blood samples, isolating the serum, and then loading (5) or (10) microliters of the serum after mixing it with loading buffer. The former amount yielded better bands on the gel but these are still so jammed that you can name them a smear rather than separate bands.

Please advice,
Regards

[admin]

It seems your antibodies are not very good (as you are getting many many bands - not good), it would help to optimize also the blotting procedure. You should take a look in the forum and this website how to optimize western blots.

Also, post the western blot picture so that we can help you more,

admin

[preethi]

Hi
We have problems with our westerns. We see a trend in our western image from left to right. There is a increase in signal in bands from left to right. We tried different antibodies and confirmed that there is no problem with antibodies. We loaded constant amount of protein, but still we see the trend. We run gels in Bio-Rad gel apparatus. Transferring is done in Semi dry Transfer gel Apparatus at 20 volts for 1hr & 15 minutes. The memberanes are developed using Femto developing solution. Can you give us suggestions,

best regards