Nanoscale

[blueangel500]

Hello,

I will be beginning to work in a lab soon and I am trying to write my experimentation procedure. I am doing everything at a nanoscale and it is confusing me, would you have any tips? My teacher recommended converting everything to moles, but I do not know if the pipettes measure moles? Would they not be using micrograms?

Also, I am trying to figure out how many experiments I can do with one batch of proteins: 100 ug molecular weight: 37kDa. I am testing for the protein presence with my test and I was wondering also what range of numbers I should test with the given amount? I read that a similar protein's detection limit is 0.1 pmol (0.1 μM in 1 μL) but I'm not exactly sure how to interpret/convert the numbers.

Thank you for your help!
Sarah

[donnahardy2]

Hi Blueangel,

You have an exciting research opportunity and you are going to be working with extremely small amounts of protein. Your teacher has given you good advice, and here is an explanation that will help you calculate the quantity of protein in your samples.

I think I need a little more information about your specific sample, so this is an example. I've done this one step at a time. Look at each step to make sure you understand the conversion and let me know if you don't understand any of the steps:

If a batch of protein is 100 ug and it is dissolved in 1 mL(1000 uL) of buffer, then the concentration is 0.1 ug/uL. (What is the volume of the batch of protein?)

To convert 1 ug/uL to moles you would multiply by:

0.1 ug/uL x 1 uM/37 ug = 0.0027 uM/uL

There are 1 million picomoles in a micromole, so to convert micromoles (uM) to picomoles:

.0027 uM/uL x 1,000,000 picomoles/micromoles = 2.7 x 104 (10 to the 4th) picomoles/uL

What is the dynamic range of your assay? You will probably want to dilute your sample to a range of say 0.1 to 100 picomoles/uL, for example. So let me know what you will do next with your samples.

Donna Hardy

[heatherL]

Hi Sarah,

It sounds like you are having trouble with conversions. Donna has already given you some great advice. I also found some sites with basic examples to help you with your calculations. If you give us more specific information, we can help you with your unit conversions.

Site about mole calculations: http://misterguch.brinkster.net/molecalculations.html
Converting moles to milliliters: http://antoine.frostburg.edu/chem/senes ... ters.shtml
Wikipedia article about concentration: http://en.wikipedia.org/wiki/Concentration

I hope this helps!

Heather

[blueangel500]

Thank you Ms.Hardy & Heather for that information!! Very helpful! Although, I am having trouble with the specific calculations.

My new protein is a 200 µl sample with a concentration of 1.34mgml-1 (or 45.0 mM) - molecular weight:29767.14. I am wondering how to use serial dilutions (or any other technique) to get it down to the levels I need? The concentrations I was considering are 25 pmol, 12.5 pmol, 6.25 pmol, 3.123 pmol, .78125 pmol, 0.290625 pmol (I just divided by two as that is what I saw in a lot of literature). I am scared that I will not be able to detect such low levels of proteins (in theory it should work, but I have never done research in a lab, so I am very nervous that I might alter something accidentally and ruin the experiment results. If this happens, will I still be able to present my project?).

The actual levels of this protein in plasma are vary from 1000 pg/mL to 1000 ng/mL. Will the concentrations I picked be low enough to correspond with the actual levels or would you recommend other concentrations, considering the limited amount of protein I have? (I am having difficulty converting the concentration to a molar amount - is there a clear method for this?)

Finally, I am a finalist for Canada's team to ISEF and we have to hand in reports and powerpoints. I have not been able to get into the lab yet as I have been raising funds for my project, so I only have about a week to actually conduct the experiments before the due date. Since ISEF is in May, do they usually allow finalists to submit what they have done at that point and continue researching? I can finish most of my reports beforehand (but I am not sure about the analysis part because I may not have the actual results yet -will be anticipated results be sufficient?)

Thank you so much for all your help!!
Sincerely,
Freaking Out

[donnahardy2]

Hi Blueangel,

You have a tremendous challenge ahead, but just try to go slowly and take one step at a time and your project will turn out fine.

First, let’s check the calculations. Your first calculation is correct.

0.134 mg/mL x mM /29767 mg = 45 nanomoles/mL or 45,000 picomoles/mL

Next, to dilute 45,000 picomole/mL to 25 pg/mL, you would need to make a 1:1800 dilution. What type of pipettes do you have? What final volume do you need of each dilution? You will probably need to make the 1:1800 dilution in two steps.

However, to answer your next question, if you want to test in the range of 1000 pg/mL to 1,000,000 pg/mL (1000 ng), and you are starting with 134,000,000 picograms/ ml you need to make a 1:134 dilution for the first sample, and then a serial two-fold dilution for the rest of the samples.

Before you continue, please verify if you want to use pg/mL or picomole/mL because it makes a big difference in the calculations. Proteins are usually used in mg/ng/pg quantities, but I need for you to verify this, because I don’t know what protein you are working with.

What type of protein are you working with? Are there any concerns about the stability of the sample?

To enter in the science fair, you definitely need to stop and write up what you have done at the deadline. After you have submitted your entry, you can continue doing your research and present the actual results you have obtained plus any new data at ISEF. However, you cannot change your abstract which has been submitted, so it would be best to have some data to present with an analysis in the original entry. Do try to generate some data before the first deadline.

Please post again soon to answer my question about the normal range of your protein.


Donna Hardy

[blueangel500]

Hi Ms. Hardy,

The statistics I was given were 1000pg/mL and 1000 ng/mL, but all research and my professor suggests converting to molar scale, therefore I hope to use 25 pmol, 12.5 pmol, 6.25 pmol, 3.123 pmol, .78125 pmol, 0.290625 pmol. How exactly do you calculate the dilutions for the samples? I will need about 1uL of each concentration. I have the data sheet but it won't let me upload. I have been warned against freezing/thawing cycles.

Thank you so much!
Sarah

[donnahardy2]

Hi Sarah,

Thanks for confirming the units that you want to use.

Going back to the last posts, we agreed that the standard that you have available is 45 nM/mL or 45,000 picomoles/mL or 45 picomoles per liter.and you want to your highest concentration to be 25 pmoles per milliliter? (Please confirm because 25 pmol could be interpreted as 25 pmolar, or 25 pmol per liter).

To dilute from 45,000 picomoles/mL to 25 picomoles/mL, you need to dilute your sample 1:1800. This dilution is too high to do in one step, so I recommend doing it in two steps:

This dilution will require a variable 10 uL pipette. Let me know if you don't have a pipette that will work for 6 uL.

1. First transfer 6 uL of the sample into a tube containing 995 uL of suitable diluent.. This is a 1:166 dilution and the concentration of the standard is now 271 picmoles/mL. Mix this dilution very well.

2. Next, transfer 10 uL into 100 uL of suitable diluent. This is a 1:10.8 dilution and will bring the concentration down to 25.1 pmol/mL. You can call it 25 pmol/mL.

There are other possible ways to do the dilution, so let me know what pipettes you have available if you need an alternative method.

Please edit your last post and delete your personal e-mail address. Science Buddies volunteers are not allowed to contact students directly. This is a rule to protect your privacy and safety.

However, I do have some other suggestions if you need more help. From your description of the project so far, it sounds like you are working with cytokines, or a similar class of molecule. Cytokines are very unstable proteins, and it is important to work quickly, keep the samples cold, and dilute into buffers that contain protective proteins when working with them. Here is a link to an instruction manual for a cytokine assay that includes lots of details for handling the samples.

Please note the dilutions of the standard recommended on page 11 of this instruction manual; it is a series of 1:4 dilutions/

http://www.bio-rad.com/webroot/web/pdf/ ... 10004F.pdf

If you are working with a kit like this, I recommend looking up the toll-free number for the technical support department of the kit manufacturer and calling to talk to someone who knows about the kit. You should be able to talk to someone who can review the dilutions that you want to make and answer all of your other questions so that you will be ready to start your experiment. I am suggesting this because I think I have explained how to do the dilution you have requested, but it seems to be a very high dilution for this type of protein.

Also, please make sure you run one standard curve as directed in the instructions of the kit you are using so you can compare your results to the expected standard curve.

Please post again if you have more questions.


Donna Hardy

[blueangel500]

Thanks so much for the information. I am a little confused as to the concentrations. For the assay, I am supposed to spot different concentrations of the protein onto the membrane (25 picomoles, 12.5 picomoles, 6.25 picomoles, etc.) Because they are in solution, I am unsure how to get it down to that amount exactly. I read that some assays use spots that are 10 uL and some are only 1. Do you have any suggestions? Also, for the control, I am limited with resources right now. Would you recommend any inexpensive proteins for controls? I have a 10% solution of Bovine Serum Albumin that I am using as the blocking buffer. Would I still be able to use that as the control?

Thanks again!!

[donnahardy2]

Hi Blue Angel,

Can you give me the catalog number and manufacturer of the kit you are using. I can check the instruction manual for the assay and give you some specific suggestions for your experiment. It is difficult without knowing exactly what assay you are doing.

Donna Hardy