• Join over 1.2 million students every month
  • Accelerate your learning by 29%
  • Unlimited access from just £6.99 per month
Page
  1. 1
    1
  2. 2
    2
  3. 3
    3
  4. 4
    4
  5. 5
    5
  6. 6
    6
  7. 7
    7
  8. 8
    8
  9. 9
    9
  10. 10
    10
  11. 11
    11
  12. 12
    12
  13. 13
    13
  14. 14
    14
  15. 15
    15
  16. 16
    16
  17. 17
    17
  18. 18
    18
  19. 19
    19
  20. 20
    20

Isolation & Characterisation of Proteins. The purpose of conducting this experiment is to study protein separation using two different methods, which are the SDS-polyacrylamide gel and ion exchange chromatography

Extracts from this document...

Introduction

Name: Ng Yen Pheng Student ID: 22353046 Day and date: Tuesday, 3 April 2012 Title: Isolation & Characterisation of Proteins Aim: The purpose of conducting this experiment is to study protein separation using two different methods, which are the SDS-polyacrylamide gel and ion exchange chromatography. This practical also aims to study what types of ion exchangers which are more suitable to separate different types of proteins. This experiment also aims to compare the separation of proteins by SDS-PAGE and ion exchange chromatography. Results: Part A: SDS-PAGE Figure 1: Image of SDS-Polyacrylamide gel electrophoresis. Table 1: Distance migrated (cm) by different length of protein fragments denatured by SDS Bands Molecular Weight, MW (kDa) Log10 (MW) Distance migrated (mm) 1 250 2.40 14 2 130 2.11 24 3 100 2.00 33 4 70 1.85 43 5 55 1.74 48 6 35 1.54 - 7 25 1.40 66 8 15 1.18 75 9 10 1.00 87 [( - ) in the table indicates that the band is missing] Figure 2: Graph of log (Molecular weight) against distance migrated for marker. Table 2: Distance migrated and size of fragments of 3% haemoglobin being denatured by SDS with different dilution factor in the presence of 1% of BSA. Dilution factor Distance migrated (mm) Log (Molecular weight) Molecular Weight (kDa) 1/5 (Sample A) 43 1.819 65.892 90 0.929 8.497 1/10 (Sample B) 44 1.780 63.083 91 0.910 8.135 1/50 (Sample C) 45 1.781 60.392 92 0.891 7.788 Calculation: To calculate molecular weight for samples Using the equation in the graph above, y = -52.836 x + 139.1 where x = log (MW) For sample A, when the protein fragment has migrated 43 mm y = 43 43 = x = = 1.819 Molecular weight = 101.819 = 65.892 Another band in sample A migrated 90 mm y = 90 90 = -52.836 x + 139.1 x = = 0.929 Molecular weight = 100.929 = 8.497 For sample B, when the protein fragment has migrated 44mm y = 44 44 ...read more.

Middle

of DEAE column, y = 0.0053x in which y = absorbance and x = albumin mass By using equation: y = 0.0053x Mass of serum albumin in eluent fraction in tube 1, �g 0.069 = 0.0053x x = = 13.02 �g/0.1ml When 0.1ml consists of 13.02 �g of serum albumin 1ml consists of = 130.19 �g Concentration of serum albumin in each eluent fraction in tube 11, g/ 100mL When the mass of BSA present is calculated to be 130.19 �g in 1ml solution Thus, concentration is = 130.19 �g/ml Same method of calculation was applied to tube 1 - 10 and included diluted sample in order to calculate mass and concentration of serum albumin in eluent fraction. Total mass of serum albumin recovered in CM column in tube 1 - 2, �g = 130.19 + 145.28 + 226.41 + 271.70 + 292.45 + 264.15 + 256.60 + 305.66 + 271.70 + 283.02 = 2447.16 �g Percentage recovery of BSA in CM column, % = [ ] � 100% = x 100% = 37.27 % Figure 8: Elution profile of mass of serum albumin against test tube in CM column. Discussion: Part A: SDS-PAGE When proteins are to be separated using electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is often chosen. SDS is a strong detergent which can denature proteins, unravelling the tertiary structure of proteins to secondary or primary (Puri, 2006). This is due to the interference to the hydrophobic interactions of proteins which act to stabilize them (Puri, 2006). SDS molecules have negative charges which can mask the proteins intrinsic charges (Puri, 2006). In SDS-PAGE, proteins denatured by SDS require different time. The 5% stacking region in the SDS-PAGE gives proteins time to be completely denatured, ensuring that the samples in all the columns have been denatured and start running in the gel at the same time. From the gel obtained in figure 1, 8 bands were found instead of 9. ...read more.

Conclusion

Of the two methods to separate proteins, IEC would yield a more accurate result as compared to SDS-PAGE. SDS-PAGE only shows bands which, for the determination of types of proteins present, it has to be referred to a protein ladder. Sometimes the bands may not be obvious enough to determine the distance migrated. Hence determination of types of proteins may not be accurate. In IEC, proteins bound to the charged resin beads to different extent according to their polarity. The results then passed to the detector to be analysed, presenting types of proteins present as peaks at particular retention time. Peaks and retention time are different for different proteins. Hence this will produce result which is much more accurate. Besides, proteins sizes may vary greatly. In electrophoresis, pore size is even throughout the gel. Proteins with smaller size may not be well separated in SDS-PAGE, whereas in IEC, proteins will be adsorbed to the resin beads to different extent, according to their polarity. Proteins which are not adsorbed onto the resin beads will be flowed out first then only the bound proteins will be "knocked off" from the resin beads. This enables better separation of proteins. Conclusion: In SDS-PAGE, the greater the concentration of sample, the slower the sample will be travelling at. Molecular weight of haemoglobin for 3 dilution factor of 5, 10 and 50 are 8.497kDa, 8.135kDa and 7.788kDa. While molecular weight for BSA in 3 different dilution factor is 65.892kDa, 63.083kDa and 60.392kDa. This indicated that high molecular weight BSA travelled slower than haemoglobin. Haemoglobin which present at 3% travels slower that BSA which present at only 1%. DEAE column is the most suitable method used to measuring haemoglobin as it has a high percentage of recovery which is 98.28% while CM column only 68.48%. However, CM column separates BSA better since it has higher percentage of recovery which is 37.27% while DEAE only 15.84%. IEC separates proteins better than SDS-PAGE does. ...read more.

The above preview is unformatted text

This student written piece of work is one of many that can be found in our University Degree Applied Biology section.

Found what you're looking for?

  • Start learning 29% faster today
  • 150,000+ documents available
  • Just £6.99 a month

Not the one? Search for your essay title...
  • Join over 1.2 million students every month
  • Accelerate your learning by 29%
  • Unlimited access from just £6.99 per month

See related essaysSee related essays

Related University Degree Applied Biology essays

  1. Isolation and Purification of Egg White Proteins

    = 0.147/0.0402 = 3.657 mg Mass of Ovalbumin (diluted 1/10) = 0.177/0.0402 = 4.403 mg Mass of Lysozyme A (diluted 1/10) = 0.085/0.0402 = 2.114 mg Mass of Lysozyme A (undiluted) = 0.064/0.0402 = 1.592 mg Mass of Lysozyme C (diluted 1/10)

  2. Bi technology and food security: The clue for a new green revolution?

    In this context, biotechnology is not the key for food security: it can be, if well managed, the key to improving yields and quality of plants and environment, but the problem to alleviate hunger is not food production but the different policies of distribution of food.

  1. This experiment was carried out to separate and characterize the protein mixture which contained ...

    The absorbance of haemoglobin in each eluent fraction of CM column Test tubes 11 12 13 14 15 16 17 18 19 20 Diluted sample Absorbance, 595nm 0.009 0.033 0.035 0.029 0.026 0.022 0.020 0.018 0.014 0.013 0.134 Concentration of haemoglobin, �g/ml 77.14 282.86 300.00 248.57 222.86 188,57 171.43 154.29

  2. Food Science -Experiments to Determine the Properties and Uses of Enzymes in Food Preperation.

    Tenderizing compounds containing various enzymes, usually proteases may be used to hydrolyze some of the proteins in meat. The enzymes include papain and mopapain from the green papaya fruit, bromelain from pineapple, ficin from figs, and actinidin from kiwifruit. The compounds are applied to the surface of meats prior cooking.

  1. Hypothesis-based Science. An example of hypothesis-based science given in the textbook is that ...

    Outside of science and other circles of higher learning, most people solve problems in irrational ways. Problems and questions are approached from many different angles, and thus you end up with just as many different conclusions. Therefore, it is important that science always adhere to doing research with the utmost care in detail and always following the scientific method.

  2. Regional anatomy - case studies of the spinal region

    anesthetics or medicines into the CSF, or injecting dye into the CSF for identifying disc problems on X-ray pictures.

  1. EFFECT OF Light wave Length on Photosynthisis

    The light independent phase also known as the dark phase, takes place in the stromal, within the chloroplast. The result of the process is the conversion of carbon dioxide into sugar. This reaction does not depend on light, however it depends on the products of the light dependent phase.

  2. Isolation & Characterisation of Proteins

    0.370 0.370 = 0.0231x + 0.0883 x = = 12.19 kb When y = 0.323 0.323 = 0.0231 + 0.0883 x = = 10.16 kb When y = 0.263 0.263 = 0.0231x + 0.0883 x = = 7.56 kb When y = 0.182 0.182 = 0.0231x + 0.0883 x =

  • Over 160,000 pieces
    of student written work
  • Annotated by
    experienced teachers
  • Ideas and feedback to
    improve your own work