Protocol Online logo
Top : New Forum Archives (2009-): : SDS-PAGE and Western Blotting

Plant isoenzymes! - (Dec/02/2009 )

Hi!

I need seriously your helps. I am working with plant enzymatique activities. Now, Im working with plant peroxidases but I have problems for see the bands in the geles (PAGE). After to incube the extract with o-dianisidina and hidrogen peroxide and put the gel under 25 C or 4 C and dark I cannot watch the bands. Someone knows what can be the problems.

Thanks :lol:

Renaldo

-biorenaldo-

hi tere i m sorry but i cud not understand the problem completely. Why dont you stain your gel with a regular conventional stain to visualize??

-Pradeep Iyer-

First question did your check if in your starting material the enzymatic activity was still present before you started the PAGE?
It wouldn't be the first time that activity is absent in the samples.

-Gerard-

(stupid question) which gel formulation are you using?

sds-page will denature your enzyme.

native page: what is the pH? does the pH fall within the active range of your enzyme?

do you replace the buffer in the gel prior to activity staining? if so, are you sure that your enzyme didn't wash out?

we require more information to properly troubleshoot your problem.

-mdfenko-

Pradeep Iyer on Dec 2 2009, 06:13 AM said:

hi tere i m sorry but i cud not understand the problem completely. Why dont you stain your gel with a regular conventional stain to visualize??


Thanks for answering me!
I work with embryiogenic callus in potato. I induce the somatic embryogenesis process and do isoenzymatiques profiles between embryogenic and non embryogenic callus. I have found activities but I cannot see bands in the gels PAGE, specifically peroxidases. I do not estimate enzimatique activities for espectrophotometry, only from gels PAGE without SDS and zymograms.
First, I estimate the concentration in proteins of the extract obtained, following the Bradford method. Second, I extract the proteins with TCA (tricloroacetic acid) for two hours at a temperature of 4 C and centrifuge during 15 minutes. I use the precipitated that I have obtained as samples to run the gels. Then I use a Laemmli buffer volume (Tris Ph 6,8, without SDS, 10 % glycerol, 0,05 % bromophenol blue) to charge 50 g - 75 g of proteins. The running buffer is glicine pH 8,3. After finishing the run, I incubate the gel in a solution of acetate buffer 0,2 M ph 5 (35 ml); 6 mM o-dianisidine (substrate) (14 ml), peroxide hydrogen 100 % (1 ml). I put the gel under environmental temperature but I do not a see a profile. I also put the gels under cold temperature (4C), dark and nothing happens. I have not yet used the purified extracts for the isoenzymatiques analyses. This might be the problem. What do you think?
I have put the sample (purified extracts and pellet proteins) under 70C conditions until I find solutions since I would like to determine others activities like esterase, acid phosphatase, leucine aminopeptidase and proteases. Does someone know protocols for determining these activities? Are there protocols for determining these activities in gels PAGEs on these website?
Thanks for your interest.
Renaldo Salazar

-biorenaldo-

Gerard on Dec 2 2009, 07:03 AM said:

First question did your check if in your starting material the enzymatic activity was still present before you started the PAGE?
It wouldn't be the first time that activity is absent in the samples.


Thanks for answering me!
I work with embryiogenic callus in potato. I induce the somatic embryogenesis process and do isoenzymatiques profiles between embryogenic and non embryogenic callus. I have found activities but I cannot see bands in the gels PAGE, specifically peroxidases. I do not estimate enzimatique activities for espectrophotometry, only from gels PAGE without SDS and zymograms.
First, I estimate the concentration in proteins of the extract obtained, following the Bradford method. Second, I extract the proteins with TCA (tricloroacetic acid) for two hours at a temperature of 4 C and centrifuge during 15 minutes. I use the precipitated that I have obtained as samples to run the gels. Then I use a Laemmli buffer volume (Tris Ph 6,8, without SDS, 10 % glycerol, 0,05 % bromophenol blue) to charge 50 g - 75 g of proteins. The running buffer is glicine pH 8,3. After finishing the run, I incubate the gel in a solution of acetate buffer 0,2 M ph 5 (35 ml); 6 mM o-dianisidine (substrate) (14 ml), peroxide hydrogen 100 % (1 ml). I put the gel under environmental temperature but I do not a see a profile. I also put the gels under cold temperature (4C), dark and nothing happens. I have not yet used the purified extracts for the isoenzymatiques analyses. This might be the problem. What do you think?
I have put the sample (purified extracts and pellet proteins) under 70C conditions until I find solutions since I would like to determine others activities like esterase, acid phosphatase, leucine aminopeptidase and proteases. Does someone know protocols for determining these activities? Are there protocols for determining these activities in gels PAGEs on these website?
Thanks for your interest.
Renaldo Salazar

-biorenaldo-

mdfenko on Dec 2 2009, 01:11 PM said:

(stupid question) which gel formulation are you using?

sds-page will denature your enzyme.

native page: what is the pH? does the pH fall within the active range of your enzyme?

do you replace the buffer in the gel prior to activity staining? if so, are you sure that your enzyme didn't wash out?

we require more information to properly troubleshoot your problem.


Thanks for answering me!
I work with embryiogenic callus in potato. I induce the somatic embryogenesis process and do isoenzymatiques profiles between embryogenic and non embryogenic callus. I have found activities but I cannot see bands in the gels PAGE, specifically peroxidases. I do not estimate enzimatique activities for espectrophotometry, only from gels PAGE without SDS and zymograms.
First, I estimate the concentration in proteins of the extract obtained, following the Bradford method. Second, I extract the proteins with TCA (tricloroacetic acid) for two hours at a temperature of 4 C and centrifuge during 15 minutes. I use the precipitated that I have obtained as samples to run the gels. Then I use a Laemmli buffer volume (Tris Ph 6,8, without SDS, 10 % glycerol, 0,05 % bromophenol blue) to charge 50 g - 75 g of proteins. The running buffer is glicine pH 8,3. After finishing the run, I incubate the gel in a solution of acetate buffer 0,2 M ph 5 (35 ml); 6 mM o-dianisidine (substrate) (14 ml), peroxide hydrogen 100 % (1 ml). I put the gel under environmental temperature but I do not a see a profile. I also put the gels under cold temperature (4C), dark and nothing happens. I have not yet used the purified extracts for the isoenzymatiques analyses. This might be the problem. What do you think?
I have put the sample (purified extracts and pellet proteins) under 70C conditions until I find solutions since I would like to determine others activities like esterase, acid phosphatase, leucine aminopeptidase and proteases. Does someone know protocols for determining these activities? Are there protocols for determining these activities in gels PAGEs on these website?
Thanks for your interest.
Renaldo

-biorenaldo-

i think that your primary problem is your method to concentrate (isolate?) your protein. tca is a protein denaturant. it may be that you can, with some proteins or under some conditions, renature the protein but i am unaware of any such procedure.

why don't you try ammonium sulfate, peg, acetone or some other precipitant that won't denature the protein.

also, some proteins don't like being frozen.

-mdfenko-