Protein quantitation guidance (SXHL288)

Transcription

1 Protein quantitation guidance (SXHL288) You may find it helpful to print this document and have it to hand as you work onscreen with the spectrophotometer. Contents 1. Introduction Protein Assay A quick refresher: overview of the protein assay technique Instructions for using the spectrophotometer... 2 Familiarisation with the spectrophotometer... 3 Collecting data from your protein standards... 3 Drawing your curve from the standards absorbance values... 3 Collecting absorbance values from your WAT and BAT samples... 4 Using your standard curve to determine the range of absorbance values in the linear range... 4 Using your standard curve to determine the protein concentration in each experimental sample Protein Calculations... 5 Calculating the protein concentration in each of the original samples before dilution... 5 Calculation of protein per mg of adipose tissue Analysing your data: comparisons between experimental groups... 6 (a) Comparison between groups: amount of protein per mg of tissue... 6 (b) Comparison between groups: amount of protein per adipose tissue depot... 6 (c) Comparison between groups: protein content per cell... 7 Appendix: Physiology laboratory spectrophotometer guidance Introduction This lab guide covers the use of the interactive onscreen spectrophotometer you will be using to measure changes in both WAT and BAT composition as a result of cold adaptation, by examining changes in one biochemical constituent of adipose tissue: proteins. The assay is carried out separately from that for lipids, although the lipid and protein extractions were conveniently made from the same tissue sample, as you will have seen in the accompanying videos. The tissue sample was homogenised in a reagent that precipitated the protein and extracted the lipid into the solvent phase. Thus, for the lipid assay you are able to use the solvent phase, while for this protein assay you use the precipitated residue. Before you start, make an educated guess about what proportion of the BAT or WAT is protein. Write down your prediction and later compare it with your results. 1 P a g e

2 2. Protein Assay 2.1 A quick refresher: overview of the protein assay technique The protein assay is based on a colorimetric analysis. The Bradford dye stains proteins, producing a dark blue colour which absorbs light at 595 nm. The absorbance of the reaction mixture at this wavelength is proportional to the concentration of protein within a limited concentration range, and it can be measured using a spectrophotometer. The assay is carried out by placing the sample (which is in a container called a cuvette) into the spectrophotometer. The spectrophotometer automatically passes a light beam through the cuvette, detects how much light has passed through the sample, and reports the absorbance value. Samples with higher protein concentrations will therefore have higher absorbance values. In order to determine the relationship between absorbance and protein concentration, you first use samples containing a known amount of protein. These samples are known as standards. Once absorbance values are collected from the standards, the relationship between concentration of protein and absorbance is drawn as a graph called a standard curve. You then can collect absorbance values for your experimental samples and use the standard curve to determine how much protein is present in each sample. You are advised to carry out your lipid study first and the lipid activity guidance notes provide more detailed instructions and guidance on using the spectrophotometer and advice on entering data in the Data Analysis area. If you are familiar with the spectrophotometer from your lipid study, you should skip the initial familiarisation steps below and proceed directly to the section titled Collecting data from your protein standards on Page Instructions for using the spectrophotometer You will access the spectrophotometer several times in your study. In Task 2 you are asked to explore the spectrophotometer In Task 4 you gather your experimental data In Task 5 you are asked to analyse your data You should use this Lab Guide to help with these tasks, and the instructions below will help you as you work through them. If you use a printed version of these notes, you might find it helpful to tick off each section as you complete that stage. If you have already carried out your lipid assays, you should be familiar enough with how the spectrophotometer is used, so you may skip the first section below. A quick guide to the spectrophotometer functions can be found in the appendix. Some guidance times are provided to help you plan the online components of this activity. 2 P a g e

3 Familiarisation with the spectrophotometer (allow up to 30 minutes) Go to the Physiology lab, navigate the lab panorama to the hotspot on the spectrophotometer image (hint: it s on the left hand bench!). Select the hotspot and read the text in the right hand panel. To access the spectrophotometer, select Launch Activity. You should launch this activity in a new tab or new window to allow you to return to the main laboratory more easily. First open the help pages by selecting the [Show Help] button. These pages explain the functions of the various controls on the interactive spectrophotometer. You can access these help pages at any stage. When you have finished reading through, you can close the pages by selecting [Hide help]. o You can access these help pages at any stage and you will find them reproduced at the end of this guide in an Appendix as a handy reference When you have finished reading through, you can close the pages by selecting [Hide help]. This spectrophotometer is used for both the lipid and protein quantitation assays: only the wavelength used and the samples differ. Collecting data from your protein standards (allow up to 1 hour) Once you have familiarised yourself with how to operate the Spectrophotometer, select the [Samples] button to open the list of samples available. From the pull-down menu, select PROTEIN CALIBRATION. Draw up a table in your lab book in which you will record your data. You should leave enough space for two readings for each of the standard samples in the protein calibration activity. Use the spectrophotometer at the appropriate wavelength (595 nm) to take the measurements to obtain values to produce your standard curve. You must set the reference value first, before taking any readings, by selecting Reference to place the reference cuvette into the Spectrophotometer. Use the R button to set the reference value. Now you are ready to take readings for your standard curve. Take absorbance readings in duplicate (2 separate readings per standard, pressing T for each reading). Record your data directly into the table in your lab notebook. When you have collected all the readings, calculate the mean value for each pair of absorbance readings for each standard concentration. Drawing your curve from the standards absorbance values (allow up to 30 minutes) A tool is provided to draw a curve from your standards. This is found in the Data Analysis area, accessed from the lab portal. You should open this in a new tab or window of your browser, for ease of use. Enter your mean values for each of the standards directly into the online table under the Standard curves tab in the Data Analysis room, having selected the Absorbance values for proteins option first. When you have entered values for all of the standards, select Plot graph to see the results plotted. Before moving onto the next section, take a look at your standard curve. Note that as the concentration of protein in the sample increases, the graph indicates that the absorbance of the sample increases. This you would predict, as the assay is based upon the interaction between the Coomassie dye and protein, and as protein concentration increases, the amount of light absorbed by the sample increases. The graph presents the relationship in the form of a line of best fit. 3 P a g e

4 If you are unfamiliar with using standard curves, try this thought experiment before moving on. Imagine you have a sample that gave an absorbance reading of at 595 nm. To use the standard curve to estimate the protein concentration in that sample, look at your standard curve and follow the horizontal line from the 0.2 point on the absorbance axis until it hits the plotted line. Now draw a vertical line down from this point to meet the protein concentration axis, and read this value. This is the concentration of protein in the sample with an absorbance of Note that the units of concentration are in µg/ml (micrograms per millilitre) and this can also be written as µg ml -1 ). Collecting absorbance values from your WAT and BAT samples (allow up to 2 hours) You should notice that, for your experimental protein samples, there are two different samples per animal. These are samples that have been diluted from the original extract. One is a 1-in-5 (1/5) dilution, the other is a 1-in-10 (1/10) dilution. These dilutions are used because the concentration of protein in a non-diluted sample is too high to be accurately determined in this assay. Draw up a table for data recording in your lab notebook. o Note that you will need to record WAT (1/5 and 1/10) and BAT (1/5 and 1/10) absorbance values (in duplicate) for each of your selected animals (5 cold and 5 warm). Obtain absorbance values for each of your test samples in duplicate (i.e. take two separate readings per sample) from the spectrophotometer. You should record readings from both 1/5 and 1/10 dilutions (thus four readings in total for each sample). Write these down in your lab notebook and calculate the mean for each pair. Using your standard curve to determine the range of absorbance values in the linear range (allow up to 30 minutes) Before using your standard curve to determine the concentration of protein in your samples, you first need to determine which of the values you have obtained (from the 1/5 or 1/10 dilutions) should be used. Recall that a standard curve is reliable only in a particular range of concentrations. A standard curve can only be used if the concentration of the sample you are analysing (or, by analogy, its absorbance) is similar to that of the standard on which the calibration was made. Even for a standard sample, there is a limited range for which the relationship between absorbance and concentration is linear, and outside this range the standard curve is unreliable. For the protein standard curve drawn up using the reagents used in this study, you can assume that relationship between absorbance and protein concentration is linear in the concentration range µg/ml. Using this, you can now determine the absorbance range that can be used to obtain reliable estimates of protein concentrations in your experimental samples. From your standard curve, what absorbance would you expect a sample to have if it contained protein at a concentration of 5 µg/ml? This value should just be an approximate reading, it doesn t have to be accurate, just an estimate (Hint: use the above instructions about drawing a line from one axis to the standard curve and across to the other axis). Record this absorbance value. What was the mean absorbance value for your 200 µg/ml protein standard? 4 P a g e

5 Write these two absorbance values down in your lab notebook as you will use these to exclude some of your experimental samples from further analysis. From the previous steps you now have a range of absorbance values that corresponds to the linear range for the protein detection assay you have used. Look through the absorbance data for each animal in your lab notebook (both 1/ 5 and 1/10 dilutions) and decide which values should now be excluded from further analysis due to them having an absorbance that is too high or too low (i.e. those that are outside the linear range of absorbance for samples containing between 5 and 200 µg/ml protein). o Now that you have determined which of your absorbance values can be used, you should decide on which set of data you will use in subsequent calculations. o It is likely that some values in the 1/5 dilution range have absorbance values too high or that some 1/10 dilutions might have a value that is too low (i.e. outside the linear range between µg/ml you have determined). o If this is the case you should exclude that entire dataset (e.g. all values for all animals from that same dilution) and continue with the dataset from the other dilution for your further calculations. If both dilutions have erroneous readings, you may have to exclude some values from your chosen dataset. Ensure that you make a note of which values you use and include these in your report. Using your standard curve to determine the protein concentration in each experimental sample (allow up to 30 minutes) Return to your standard curve and enter each mean absorbance value for each of your samples that are within the linear range into the area under the table Please enter a value for A595 (arbitrary units). You will be provided with a reading from the standard curve for each mean absorbance value converted into protein concentration (in µg/ml) that you should record in your lab notebook. These are the values you will now be using. You should now have a value for the concentration of protein in each of the experimental sample dilutions that you analysed. 2.3 Protein Calculations Once you have obtained all your measurements from the spectrophotometer, produced your standard curve, and used it to measure the protein concentrations of all of your experimental diluted-samples, there are some further calculations to perform that will allow you to make comparisons between depots and animals. Calculating the protein concentration in each of the original samples before dilution Before proceeding you need an adjustment to allow you to make valid comparisons between samples and animals. This is to determine the concentration of protein in the original extract. [Remember that the protein was precipitated from this extract and the pellet was solubilised in 1 ml of NaOH solution. This was then diluted (either 1/5 or 1/10) to create the samples you assayed in the spectrophotometer.] For each sample, note the dilution factor that was used to prepare it (either 1 in 5 or 1 in 10) from the 1 ml used to re-suspend the pellet. 5 P a g e

6 Multiply the estimate of protein concentration obtained from the calibration curve for each sample by the dilution factor used and note this down in your lab notebook (i.e. for values obtained from a 1 in 5 dilution, multiply by 5; for 1 in 10, multiply by 10). You now have estimates for the concentration of protein in the 1 ml (of 1 mol l -1 NaOH) used to solubilise the protein pellet. Enter your protein data into the appropriate protein (cold or warm) tables under the Results Tab. Once the table is complete click on the Show statistics button to obtain mean protein concentrations, the SD and SEM values for each group of animals (control warm and coldadapted) and each tissue (WAT and BAT). o Note: if you notice an inputting error and alter any values, you must click on show statistics again to update the values. Make a print out of your final tables with their statistics to stick into your lab notebook. Calculation of protein per mg of adipose tissue The concentration of protein that you have just obtained from your standard curve is that of the tissue extract used (not the whole tissue sample). Recall this tissue extract was made from 30 mg of dissected adipose material. The protein was then precipitated and dissolved in a volume of 1 ml. To determine the concentration of protein per mg of adipose tissue, you now need to convert your original estimates. o Divide each of your protein concentrations by 30. o This will give you the protein concentration in µg protein (mg tissue) -1 o Keep a record of your workings and answers in your lab book. You will use these values in the following calculations. 3 Analysing your data: comparisons between experimental groups You now need to collate your data so that you can make suitable comparisons between your experimental groups. To do this, first organise them into their separate tissue groups i.e. BAT or WAT, and then further separate them by experimental groups i.e. control (warm) or cold-adapted. You can then compare groups in two ways, as detailed below. (a) Comparison between groups: amount of protein per mg of tissue For each experimental group and tissue, you need to arrange your data into the appropriate groups and then for each group calculate the mean value for protein content and calculate the standard error of the mean (SEM). SEM = Standard Deviation divided by the square root of n (where n is your sample size). You may make use of the appropriate table under the Results tab to do this or use another method. Present your results as a table or graph. To perform statistical analysis of your results, you may make use of the t-test. (b) Comparison between groups: amount of protein per adipose tissue depot You also have the masses of the adipose tissue depots that were removed from each rat. Use your results obtained so far (from Section 3a above) and the adipose tissue depot masses (from the Results tab in the online Data Analysis) to produce a comparison between groups. Remember that to convert from g to mg you should multiply by 1000 (e.g g x 1000 = 900 mg). 6 P a g e

7 So the steps to follow are: Convert the depot masses into appropriate units. Multiply each mass of depot by its protein per mg of tissue value used in (a). Make sure to use the correct units with your final values. Carry out a comparison between groups. Do your results still show the same pattern as the comparison in (a)? Look back at the prediction about the proportion of WAT and BAT made up of protein that you wrote down in your lab notebook before carrying out this activity. How does your prediction compare with the results that you have obtained? (c) Comparison between groups: protein content per cell Once you have determined the protein content per mg for each tissue sample (in step a, above), you can use your data obtained from the histological analysis of BAT and WAT tissues to calculate the protein content per cell in BAT and WAT. If you know how much protein is contained within one mg of tissue and you know how many cells are in that mg of tissue, then dividing the amount of protein by the number of cells will give you an estimate of how much protein is contained per cell. Record your calculations and results (with appropriate units) in your lab notebook. Now do a comparison between groups, as before. Do your results still show the same pattern as in (a) and (b)? 7 P a g e

8 Appendix: Physiology laboratory spectrophotometer guidance These instructions are available from within the spectrophotometer s Help pages For Protein data collection, remember to use the wavelength of 595 nm 8 P a g e