Lab report

Introduction:

Aims of experiment: In the experiment we aimed to isolate and identify specific proteins from a mixture of proteins. Due to the nature of proteins there are many ways to isolate individual proteins, during this experiment we used two purification techniques- size exclusion chromatography and ion exchange chromatography to separate our proteins and then SDS-PAGE to analyse and identify our purified samples.

Size exclusion chromatography (SEC): By using SEC you can determine reasonably quickly and easily the average molecular weight (MW) and molecular weight distribution (MWD). SEC separates molecules based on their size, this compares to Ion exchange chromatography which separates molecules based on their interactions. SEC is a technique often for the purification and analysis of both synthetic and biological polymers, in this case proteins. (1) In our size exclusion chromatography Sephadex was used, Sephadex is a combination of two other types of gel- Sephacryl and Sepharose. The positives of Sephadex include that it runs under the force of gravity and requires less packing down (2). Sephadex is beaded, large molecules pass through the gel far more easily than small molecules as they do not get stuck in the porous beads. By taking fractions of the volume that has run through the gel you can analysis the contents of the sample that initially went into the gel and thus alongside other techniques determine what proteins our sample contained.

Image: http://cnx.org/contents/ba27839d-5042-4a40-afcf-c0e6e39fb454@20.16:25/Physical_Methods_in_Chemistry_

Ion exchange chromatography: This works by separating different proteins based on their charge, different proteins have different charges due to the different R groups on the amino acids that make up their primary structure. There are different types of resin that you can use in ion exchange

chromatography depending on what the pH of the proteins you’re looking at is. Ion exchange resin is a thick liquid in which the proteins bind by electrostatic forces to the clusters of charge groups within the resin (3). If a protein has a generally positive charge it will be attracted to the cation exchanger and if a negative charge is possessed by the protein it will be attracted to the anion exchanger. To elute the molecules from the column an increasing salt gradient is used, at a low salt concentration the molecules with the weakest ionic interaction will leave the column first, by then increasing the salt concentration you elute the molecules with a higher ionic biding capacity (4). Similarly to size exclusion chromatography you take fractions of the elute and use different techniques to study the proteins within your sample.

SDS-PAGE: Proteins have a primary, secondary, tertiary and quaternary structure, when incubated with SDS (sodium dodecyl sulphate) the protein loses its secondary, tertiary and quaternary structures, this means that the protein will break into its subunits and form strands. Due to the nature of SDS the proteins will all gain a negative charge, regardless of whether they were positively or negatively charged prior to incubation with SDS (5). The PAGE (Polyacrylamide gel electrophoresis) is a process which separates the different proteins by size, the proteins are put in channels in a polyacrylamide gel and then the electric field is turned on. The electric field has a positive end towards which the negatively charged molecules are drawn and a negatively charged end which pushes the molecules away. The polyacrylamide gel is not solid but made up of lots of tunnels which pass through the whole gel, after a set time the small molecules will have got further through the gel than large molecules which will be unable to travel very far through the gel before getting stuck.

Image: http://biolympiads.blogspot.co.uk/2014/09/sds-page-tutorial.html

Pre-lab questions:

Protein Native MW (kDa)

Subunit MW (kDa)

pI Approx.

A410:A280

Bovine serum albumin (BSA) 66.5 Heavy: 30

Light: 11-12

4.6 0

Cytochrome c (horse heart) 12.3 10.6

4

-Lactalbumin (bovine milk) 14.2 4.8 0

Myoglobin (horse heart) 17 8.2 4

Haemoglobin (bovine) 64 16 6.5 4

Ribonuclease A (bovine pancreas)

13.7 9.2 0

Catalase (bovine liver) 250 60 5.1 1

Ferritin (horse spleen) 440 Heavy: 21

Light: 19

4.9 0.2

Materials and Methods:

During the experiment we followed the method detailed in the BIO2090 practical schedule (pages 5-7). During the size exclusion chromatography our void volume was 13.75mL, after this we took 35 1mL fractions in 1.5 mL Eppendorf tubes, we didn’t need to take 50 fractions as advised in the practical schedule as our eluant returned to the baseline level. During the ion exchange chromatography we took 30 1mL fractions to elute the first protein with buffer A, we then added buffer B, however 30 fractions was not enough to create another peak with the platereader so we continued and took a total of 46 fractions with buffer B present to elute our second protein. In the SDS-PAGE we loaded 2 columns with samples from ion exchange, we took the peak at fraction 20 and it was mixed with fraction 19 and 21 to create the sample for the SDS-PAGE and fraction 70 was mixed with fractions 9 and 71 to create the other sample. We only loaded our large peak from size exclusion into the SDS gel for this we used fraction 23.

Results:

Our results from putting our ion exchange fractions in a plate reader show two peaks at 280nm and one larger peak at 410nm. The first peak from 280nm and the peak for 410nm was at fraction 20, it had a reading of 0.942 at 280nm and 3.276 at 410nm. The second peak with 280nm was at fraction

70, it had a reading of 0.776. Table 1 shows the results from putting our fractions in the plate reader at both 280nm and 410nm. Figure 1 is a graph that depicts the information from the plate reader in a more accessible view; it shows the difference in the size of the peak at fraction 20 between 280nm and 410nm by plotting absorbance against fraction.

TABLE 1:

Number of Fraction Absorption Absorption(at 280nm) (at 410nm)

1 0.047 0.0272 0.047 0.0273 0.047 0.0274 0.047 0.0275 0.047 0.0276 0.046 0.0277 0.045 0.0268 0.046 0.0279 0.046 0.02710 0.047 0.02711 0.048 0.02712 0.046 0.02713 0.045 0.02714 0.046 0.02715 0.048 0.02816 0.046 0.02717 0.057 0.07818 0.23 0.88719 0.683 2.80120 0.942 3.27621 0.813 3.15122 0.37 1.53923 0.16 0.55524 0.103 0.23725 0.072 0.14526 0.066 0.11227 0.056 0.07328 0.049 0.04429 0.051 0.03530 0.058 0.03831 0.057 0.03732 0.056 0.03633 0.066 0.04234 0.061 0.03935 0.055 0.03536 0.062 0.04937 0.064 0.0438 0.051 0.03339 0.057 0.03740 0.051 0.03341 0.055 0.03742 0.062 0.04143 0.057 0.03644 0.057 0.03645 0.068 0.04246 0.055 0.03647 0.056 0.03648 0.057 0.03649 0.057 0.03750 0.06 0.03951 0.058 0.03852 0.047 0.031

BUFFER B ADDED

FIGURE 1:

During our size exclusion column chromatography we found again two peaks at 280nm, the first of these was smaller with a peak of 0.206 at fraction 9, the second higher at 0.812 at fraction 23. There was again only one peak at 410nm, this was also at fraction 23 with a peak of 1.905.The ratio of A280:A410 was at its highest at fraction 27 after the peaks of 280 and 410 at fraction 23.

Table 2 shows the information from the plate reader and the A280:A410 ratio. Figure 2 shows a graph which plots absorbance against fractions.

TABLE 2:

Fraction Number 280 nm absorbance 410 nm absorbance A280:A4101 0.054 0.028 0.51852 0.054 0.028 0.51853 0.086 0.032 0.41864 0.159 0.036 0.22645 0.165 0.036 0.21826 0.157 0.033 0.21027 0.176 0.033 0.18758 0.201 0.035 0.17419 0.206 0.032 0.155310 0.184 0.031 0.168511 0.150 0.030 0.212 0.126 0.030 0.2381

13 0.108 0.030 0.277814 0.106 0.034 0.320815 0.124 0.041 0.330616 0.187 0.090 0.481317 0.291 0.212 0.728518 0.436 0.444 1.018319 0.588 0.773 1.314620 0.713 1.151 1.614321 0.808 1.540 1.905922 0.812 1.761 2.168723 0.779 1.905 2.445424 0.691 1.865 2.69925 0.577 1.676 2.904726 0.461 1.404 3.045627 0.346 1.086 3.138728 0.257 0.804 3.128429 0.190 0.571 3.005330 0.145 0.385 2.655231 0.106 0.249 2.349132 0.081 0.154 1.901233 0.069 0.103 1.492834 0.060 0.070 1.166735 0.056 0.053 0.9464

FIGURE 2:

Figure 3 shows our run of SDS-PAGE, I have labelled the marker proteins with their length in mm and the corresponding protein in the tricine marker sample. The length of the gel was 163mm, the largest protein, Myosin (210kDa) travelled 30mm, and the smallest protein Insulin B Chain (4kDa) travelled 161mm. From this we worked out the R(BPB) for each protein and the log(MW) this is shown in Table 3, using this I plotted a graph of R(BPB) against log(MW), this is shown in Table 3. Using excel I plotted a line of best fit with the equation y = -1.8711x + 2.4916, this is shown in figure 4. Having measured the running length of the 3 unknown protiens I was able to work out their R(BPB), using the equation from the line of best fit I could then work out their log(MW) and from this their MW which came to 46.2 for protein 1, 5.88 for protein 2 and 4.4 for protein 3, this data is shown in Table 4.

FIGURE 3:

TABLE 3:

FIGURE 4:

TABLE 4:

Distance on photo mm

R(BPB) Log(MW) MW

Protein 1 72 0.441718 1.665101 46.2Protein 2 150 0.920245 0.769729 5.88Protein 3 161 0.98773 0.643458 4.4

Discussion: Discuss what happened on each column and how effective the separation of the three proteins was. Using all of the data that you have obtained, present an argument to identify which the three proteins in your sample were, and in what order they eluted from the columns. The usual error that students make is to look at the apparent masses that they obtained from SDS-PAGE, treat these as Gospel, and to not interpret them in the light of the other data. Consider the principles of how they were separated from each other, and discuss sources of error. Consider why we took

Marker Band kDa

Distance on photo (mm)

R(BPB) log(MW)

210 30 0.184049 2.322219105 37 0.226994 2.02118978 43 0.263804 1.89209555 59 0.361963 1.74036345 74 0.453988 1.65321335 88 0.539877 1.54406817 107 0.656442 1.23044916 109 0.668712 1.204127 151 0.92638 0.8450984 161 0.98773 0.60206

measurements of the samples at two wavelengths, and what feature of the protein we measuring at each wavelength.

Our ion exchange and size exclusion columns were both successful, we gained two peaks for each at 280nm and one peak for each at 410nm.

Conclusion: Identify your proteins and state how they were separated.

References: As appropriate.

1) https://www.princeton.edu/~achaney/tmve/wiki100k/docs/ Size_exclusion_chromatography.html

2) Dr Shaws lecture 3 slide 10.3) Dr Shaw lecure 3 slide 244) http://www.separations.us.tosohbioscience.com/ServiceSupport/TechSupport/

ResourceCenter/PrinciplesofChromatography/IonExchange5) http://www.bio.davidson.edu/genomics/method/SDSPAGE/SDSPAGE.html 6)