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What would happen if we don't use protease inhibitors? - for cell lysis by RIPA, NP40 or any other lysis buffer (Oct/08/2008 )

I just noticed that I can't detect the proteins of my interest in my cell lysates anymore. I used to be able to detect them even when i had 3ug/ul total protein after lysis by RIPA, but now I can not detect anything. my proteins of interest are around 20 KDa.I did Western and ELISA, but there is nothing there.

I also noticed that my SDS-PAGE bands are different from my friends' gel bands....they have many bands below 50KDa but I don't.

I use Roche's Complete mini Tablet protease inhibitors....I put 1 tablet for every 10ml RIPA

my SDS-PAGE bands are very blurry compared to my friends' bands which are very sharp.

Is my protease inhibitor expired?

-Curtis-

Hi!

The use of protease inhibitors and collection of cell lysate on ice is to avoid protein degradation. I would use them absolutely.
Redo your RIPA, use the appropiate amount of buffer according to cell number, rotate 30min, 4ºC and centrifugate 12000rpm, 5min to discard insoluble matherial.

I don't know about ELISA, but in Western you should check the pH (crucial!! tested!!) and composition of transfer solution (changing MeOH proportions you favour the migration of biger/smaller prot).

Also check you're working in reducing conditions and your protein is not in a complex with others (increasing its molecular weight).

About protease inhibitor, tablets are tricky because should be used once and then discarded to prepare fresh ones. I prefer to buy them separately and prepare in small aliquots.

I hope it helps


-Estersan-

QUOTE (Estersan @ Oct 8 2008, 02:29 PM)
Hi!

The use of protease inhibitors and collection of cell lysate on ice is to avoid protein degradation. I would use them absolutely.
Redo your RIPA, use the appropiate amount of buffer according to cell number, rotate 30min, 4ºC and centrifugate 12000rpm, 5min to discard insoluble matherial.

I don't know about ELISA, but in Western you should check the pH (crucial!! tested!!) and composition of transfer solution (changing MeOH proportions you favour the migration of biger/smaller prot).

Also check you're working in reducing conditions and your protein is not in a complex with others (increasing its molecular weight).

About protease inhibitor, tablets are tricky because should be used once and then discarded to prepare fresh ones. I prefer to buy them separately and prepare in small aliquots.

I hope it helps


thank you,
what is the total protein concentration that you get after lysis?

do proteases degrade 20KDa proteins? what is their main target?

-Curtis-

QUOTE (Curtis @ Oct 8 2008, 01:13 PM)
I just noticed that I can't detect the proteins of my interest in my cell lysates anymore. I used to be able to detect them even when i had 3ug/ul total protein after lysis by RIPA, but now I can not detect anything. my proteins of interest are around 20 KDa.I did Western and ELISA, but there is nothing there.

I also noticed that my SDS-PAGE bands are different from my friends' gel bands....they have many bands below 50KDa but I don't.

I use Roche's Complete mini Tablet protease inhibitors....I put 1 tablet for every 10ml RIPA

my SDS-PAGE bands are very blurry compared to my friends' bands which are very sharp.

Is my protease inhibitor expired?


the massive the problem with proteolysis the less high molecular mass polypeptides are to detect; from your CBB stains I do not think in terms of proteolysis; however, some polypeptides appear to be more sensitive towards others which can hardly be discriminated in a CBB gel;

maybe too long blotting time/ to much current and blotting through of parts of your p20?

-The Bearer-

QUOTE (Curtis @ Oct 9 2008, 06:05 AM)
do proteases degrade 20KDa proteins? what is their main target?


Erm... the main targets of proteases are peptide bonds???? I'm not sure by what mechanism you think a protease would selective degrage proteins of only a certain molecular weight. Difference proteases may have different specific peptide bond specificity (e.g. serine proteases), but it doesn't matter what size the total protein is. Didn't you learn this in biochemistry?

How much protein are you loading for your SDS-PAGE compared to your friend? Also, what is supposed to be the difference between the two lanes in the gel picture you showed?

-Ginger Spice-

QUOTE (Ginger Spice @ Oct 9 2008, 03:12 AM)
QUOTE (Curtis @ Oct 9 2008, 06:05 AM)
do proteases degrade 20KDa proteins? what is their main target?


Erm... the main targets of proteases are peptide bonds???? I'm not sure by what mechanism you think a protease would selective degrage proteins of only a certain molecular weight. Difference proteases may have different specific peptide bond specificity (e.g. serine proteases), but it doesn't matter what size the total protein is. Didn't you learn this in biochemistry?

How much protein are you loading for your SDS-PAGE compared to your friend? Also, what is supposed to be the difference between the two lanes in the gel picture you showed?


uh come on,....of course i didn't mean they exactly target 20KDa proteins....what i'm trying to say is that because a 20KDa protein is a small size protein then it might get degraded faster.

I load 30ug in each lane. not a particular difference, they are lysates from different samples.

-Curtis-

I would think you should check your resolving gel buffer. One of my colleagues experienced blurry small fragment bands in her gel before. Later, she found that it was the problem with her resolving gel buffer. If I am not mistaken, resolving gel buffer contained SDS can only last for a month. smile.gif

-virus_fan-

QUOTE (virus_fan @ Oct 10 2008, 03:33 AM)
I would think you should check your resolving gel buffer. One of my colleagues experienced blurry small fragment bands in her gel before. Later, she found that it was the problem with her resolving gel buffer. If I am not mistaken, resolving gel buffer contained SDS can only last for a month. smile.gif


yes, that would be a big possibility, thanks

-Curtis-

QUOTE (virus_fan @ Oct 10 2008, 03:33 AM)
I would think you should check your resolving gel buffer. One of my colleagues experienced blurry small fragment bands in her gel before. Later, she found that it was the problem with her resolving gel buffer. If I am not mistaken, resolving gel buffer contained SDS can only last for a month. smile.gif


guys....today I and my friend ran our samples on one gel to compare the samples...my samples were all blurry whereas her samples were all clear and had sharp bands....there must be something wrong with my lysis buffer or method.

and as I mentioned before, my samples get even more blurry below 50 KDa....

-Curtis-