how to prepare 200 mM HEPES/KOH pH 7.8 please help me - it is also containing 3 mM EDTANa2.2H2O, 3 mM magnesium acetate, 10 mM dithiothr (Apr/27/2006 )

Hi every body

My thesis is about extracting protein from plants. I found an article about the soluble protein extraction. And it was written that 200 mM, pH7.8 HEPES/KOH, containing 3 mM EDTANa2.2H2O, 3 mM magnesium acetate, 10 mM dithiothreitol (DTT), and 1% (w/v) polyvinyl-pyrrolidone (PVP) were used as the chemical reagents in this method. However, the authors didnt say about the volume of the reagent they used, or the procedure of this methods .

So, if somebody has ever done something like this or have some information about the preparation of this method. Please post ur info in this topic or send to my email at varisara_bo@hotmail.com. I really need it in urgent. Thanks for ur help and have a nice day

Ask the author, I often wrote emails to the author of a paper and got friendly answers

hi,
u have given the final concentrations, then u can prepare as much as u want right?
i donot see any problem in preparing this reagent?

could you be more precise about volume?
u mean
the volume of reagent which shud be prepared? OR
the volume which u shud use for ur reaction?
if this is the case, it depends on number of cells or amount of protein u will keep in this solution right!!!!

best regards


However, the authors didnt say about the volume of the reagent they used, or the procedure of this methods .

So, if somebody has ever done something like this or have some information about the preparation of this method. Please post ur info in this topic or send to my email at varisara_bo@hotmail.com. I really need it in urgent. Thanks for ur help and have a nice day
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Hi payeli,
sorry for my unclear english, eieieiei. And thanks for ur help.

The volume i meant was that the used volume of buffer per gram, or something like that, of the disintegrated plants . The author didnt say about this. So how can I know how much volume of the buffer I should use to dissolve the certain amount of dried plants.

I also wrote to the author, but so far, no reply yet.

hi kaewjung ! there i snothing wrong with what the authors have mentioned. simply prepare a extraction buffer in excess say 100ml and you can use this extraction solution in minimum quantity so as to sufficiently extract enough protein with out too much diluting your protein (the amount to be used will depend on your sample size. it is you who have to decide the amount and standardize it so that you get enough protein).
i would suggest you to prepare stock solution of all the ingredients and mix appropriately to get the working concentration ( working concentrattions are the concentrations you finally use and they are the one you mentioned ).
all the best

just one more question,

after these procedure, is it necessary to desalt the protein solution before measuring with bradford method??? I've heard somewhere that if the concentration of extracting buffer is too high, then the solution should be desalted.
in my case, it is 200 mM HEPES-KOH, is it too high?? or it is not necessary to do it

the solution you are using has salts in mM concentration and i dont think they will cause any sort of trouble in bradford assay.

hi,
u donot have to worry too much about salt conc in this case, just before protein conc estimation, dilute ur few micro litre of protein either 1:2 or 1:5 and check the conc (salts got diluted wat is ur problem then ).

gud luk
payeli

Hey guys
I tried to prepare the buffer with the same concentration as I've posted before. But it seemed like POlyvinyl pyrrolidone was unsoluble in the buffer ( I used 111,1 MW). I've checked its solubility, and it was unsoluble. I used Tris Buffer pH 7.8. Do u know how exactly to prepare this PVPP. Maybe I chose the wrong PVPP for buffer? Really have no idea. Iam in urgent as well. gotta submit the report to my professor this tuesdayT_T
Thanks for ur help