I need some help…
March 16, 2012 at 3:34 am #16227fubarioParticipant
A bit of background (NOTE you can skip to the bottom if you want, main questions lie there).
I am a 17-year old student who is having trouble in making a good lab design for enzymes (I chose lactase since the teacher told me it was relatively fast and easier to handle) having trouble in terms of finding an ‘easy’ way to assay.
I decided to make my design and research question focus around this possible idea: How do varying concentrations of heavy metal ions mixed with lactase affect the enzymes activity?
Obviously the responding variable would be the concentration of glucose and galatactose
(Inspired by this lab http://www.pjoes.com/pdf/11.3/251-254.pdf)
Now my problem is that in my school, I lack the proper resources to do a good assay (that is why i switched from pepsin to lactase) and after doing some surfing on the web for good assays, I came across these:
"A STANDARD ASSAY MIXTURE for this experiment contains the components listed below. You will be making this solution several times for each part of the experiment.
1.0 mL of lactose stock solution (100 mg/mL)
1.0 mI, of pH 7.0 buffer
2.0 mL of distilled water
A) MAKING THE ENZYME SOLUTION
Put 25 drops of Lactaid® into 25 mL of distilled water. Mix the solution by inversion several times and store it on ice until needed."
"Assay Method: A chromogenic substrate ONPG (o-Nitrophenyl-b-D-galactopyranoside) is used for the assay.
3.0ml of the substrate solution which contains 200 mg of ONPG per 100 ml of 0.1 M McIlvaine buffer, pH 4.5, is added to 1.0 ml of diluted enzyme solution. After 10 min. incubation of 30°C, reaction is stopped by adding 1 ml of 1M Na2CO3 solution. Then the absorbance at 420nm is measured with a spectrophotometer."
(http://www.mpbio.com/detailed_info.php? … ountry=223)
Now here is my problem: I am only in bio 35 and I have no clue as to how to actually prepare the assay solution even though the instructions are there, and as to how to use the assay solution.
For example, for this lab I wanna keep the amount of substrate constant, the amount of pH constant, and the amount of enzymes constant. For one particular method (http://biology.clc.uc.edu/fankhauser/La … .Oct09.pdf) it says to grind up and suspend 100 units (or mg)/mL. I want to do this, except I have no clue how to actually achieve this or do this (for the last link, I got confused at the 9000 vs 3000 unit tablets, I assume that means 9 grams versus 3 gram tablets of lactase).
ALSO IMPORTANT: As I stated earlier my school is very limited in resources, but for my lab I kind of want to attain lots of quantifiable (and maybe some qualitative) data as to the enzymatic activity (so I can do a chi-square and t-test; I would also do 10 trials for 6 different concentrations of heavy metal ions (aluminum ions)).
This leads to the overall arching question: is there a simple way to assay the enzyme that does not use spectrophotometry (do not know how to use and I also do not think my school has this) or obscure reagents (for example, when I was researching assays for pepsin, i came across several techniques that required certain regeants like the follin-reagent, which our school does not have) AND how do I prepare the reagents required (I would like to make a 100 mg/mL solution) (an example would be the assay here: http://capital2.capital.edu/faculty/wbecktel/Enzyme.htm ; this one looks particularly easier than the others).
There is also another problem, in terms of my research question, what is a compound that contains aluminum that when dissolved in one of these reagent or enzyme solutions does not react with the reagent itself (or will it even react?) and also just to clarify something: should I use the lactase solution immediately or can I wait a couple of minutes (I remember reading on one of these sites that it should be used quickly because it is unstable in certain reagents).
After this long post, i sort of feel like I understand more and that its not so bad of a problem but I would still like to clarify as to how to make a 100 mg/mL solution. I also do not know how to accurately record the enzymes activity other than by looking at the glucose and galactose concentration in a given period of time (in this case, 5 minutes); now the problem lies in what are some precise ways to measure the glucose concentration or galactose concentration for an assay mixture? ALSO I just figured out that I do not know what an appropriate concentration of heavy metal ions is for this particular experiment (just need to figure out the maximum amount of heavy metal ions per assay mixture)?
Sorry for the long post and if I came off somewhat incoherent but I suffer from a lack of understanding about most of this knowledge.
Thanks for any responses or feedback!
March 16, 2012 at 2:49 pm #110167JackBeanParticipantquote fubario:
Who does not?
First of all, you are probably confused with qualitative and quantitative. Qualitative tells you only about the quality (present/absent), while quantitative tells you how much you have, so quantitative is better than qualitative.
Anyway, if I understand you correctly, you don’t know, how to pipette your samples?
March 17, 2012 at 8:50 pm #110175fubarioParticipant
After fridays class, I ended up figuring out my lab and modified it slightly, now my my main question is 5 g/L too high of an amount of aluminum to use for 2500 mg/25 mL of lactase?
March 18, 2012 at 6:42 pm #110188JackBeanParticipant
I guess it’s 0.5% of some aluminium salt, right? So the amount of aluminium will be somewhat smaller. You can try 😉 For the first time, use some wider range (e.g. 0.01 – 10%) and after you will see the results, you can measure it more accurately for smaller range of your interest.
Once again, you can check Brenda database http://www.brenda-enzymes.info/ for some hint on inhibitor concentrations.
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