Search found 24 matches

by piefke
Fri Jul 23, 2010 12:46 pm
Forum: Molecular Biology
Topic: enzyme assay with thioredoxin
Replies: 2
Views: 2395

enzyme assay with thioredoxin

I want to test the influence of thioredoxin on the activity of my heterologously expressed enzyme. I incubate the enzyme, thioredoxin (3µM) and DTT (10 mM) for 15 minutes and then start the activity measurement. one time it worked well and I could detect an increasing activity with increasing concen...
by piefke
Fri May 21, 2010 10:45 am
Forum: Molecular Biology
Topic: tyrosine solution
Replies: 3
Views: 3950

But don´t you think DMSO will disturb the activity of my enzyme?
by piefke
Fri May 21, 2010 9:42 am
Forum: Molecular Biology
Topic: tyrosine solution
Replies: 3
Views: 3950

tyrosine solution

Hi I want to do an enzyme assay in EPPS buffer pH= 7. I want to test the effect of tyrosine on the activity of my enzyme and therefore I have to add tyrosine in different concentrations to the buffer (end concentration in 100 µl assay 10 - 250 µM). The problem is: Tyrosine is not very soluble in wat...
by piefke
Mon Dec 14, 2009 12:06 pm
Forum: Physiology
Topic: minor/major Amino acids
Replies: 5
Views: 7778

minor/major Amino acids

Hi everybody,

sometimes the amino acids are divided into the minor and major AA´s. But I don´t find any definition for minor and major amino acids... What are they?? :?
by piefke
Wed Oct 28, 2009 12:13 pm
Forum: Molecular Biology
Topic: protein gel
Replies: 1
Views: 1099

protein gel

Hi everybody!

I want to check the cleavage of a protein. The fragments I expect are 54 kDa and 8 kDa...
8 kDa is very small :( Is it possible to see this small thing on a sds-gel??? :shock:
by piefke
Wed Sep 30, 2009 1:44 pm
Forum: Molecular Biology
Topic: Thioredoxin
Replies: 2
Views: 1269

Thioredoxin

Hi!

I need thioredoxin from plant or cyanobacteria for an enzyme assay. Does anybody work with such TRX? Where did you get it from? I only found TRX from E. coli...
by piefke
Fri Jun 27, 2008 9:34 am
Forum: Molecular Biology
Topic: experiences with ni-affinity protein purification??
Replies: 4
Views: 11016

experiences with ni-affinity protein purification??

Hi! I´m trying to purify a his-tagged protein with nickel-columns. When I use imidazol in the elution buffer my enzyme is inactive after purification. I showed that imidazol is the reason for this loss of function. Because before purification I can clearly detect activity. When I use the same assay ...
by piefke
Sun May 04, 2008 9:23 am
Forum: Molecular Biology
Topic: inactive enzyme after purification with Ni-TED
Replies: 3
Views: 4186

Re: inactive enzyme after purification with Ni-TED

You helped me a lot. I´m writing my diploma thesis and now I can explain why it did not work up to now. And I will go on working with the enzyme in my pHD. So I will change the buffer next I think...
Thank you!! :D
by piefke
Sat May 03, 2008 5:24 pm
Forum: Molecular Biology
Topic: dimer-homodimer-heterodimer
Replies: 2
Views: 4516

Re: dimer-homodimer-heterodimer

:D Thank you!! You helped me a lot!!!
by piefke
Fri May 02, 2008 2:58 pm
Forum: Molecular Biology
Topic: inactive enzyme after purification with Ni-TED
Replies: 3
Views: 4186

inactive enzyme after purification with Ni-TED

Hi everybody I expressed a 54 kDa protein in E.coli. Before the purification with Ni-TED the enzyme is active. After the purification there is no activity left. What can be the reason? It is a metalloenzyme and needs Mn -ions for its activity. Could it be that the imidazole in the buffers while puri...
by piefke
Fri May 02, 2008 2:57 pm
Forum: Molecular Biology
Topic: dimer-homodimer-heterodimer
Replies: 2
Views: 4516

dimer-homodimer-heterodimer

hi

i have some questions in generall.

how do you find out if an enzyme is a dimer, and if it is a homo- or heterodimer? perhaps it is a stupid question, I don´t know...

and does it have some interesting consequences for its function if it is an homodimer or a heterodimer?
by piefke
Wed Apr 16, 2008 1:09 pm
Forum: Molecular Biology
Topic: protein purification Ni-NTA
Replies: 1
Views: 3109

protein purification Ni-NTA

Hi I´ve one little question. I purified an enzyme with Ni-NTA-Coloumns. And it worked, but I always have some bigger proteins running a little bit above my target-protein on the gel. I now have to explain this in my diploma thesis. I would understand if there would always be some smaller ones. then ...