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Immunoglobulin G

发布时间:2017-04-06
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Results

Purifying IgG from rabbit serum

Immunoglobulin G (IgG) was purified from rabbit serum under acidic conditions, achieved by using octanoic acid. Subsequent centrifugation allowed for the purified IgG present in the supernatant to be extracted, and the protein concentration to be determined via the Bradford protein assay.

Analysis of rabbit serum and purified IgG by agarose gel electrophoresis

Following the analysis of the rabbit serum and purified IgG by gel electrophoresis, the gel was subsequently stained with Coomassie brilliant blue, allowing protein detection. However, deep staining of the gel prevented the visualisation of the proteins (see ). Such a problem was prevalent in the group and may have been caused by incorrect staining and destaining. (supplied by Dr Sobia Kauser) demonstrates how the gel should have looked, illustrating the presence of proteins.

Bands are clearly present at the origin () signifying the presence of γ-globulins, which have a neutral net charge (isoelectric point) and thereby remain at the source under an electric field. Several bands, however, can be observed at the adjacent well, demonstrating the presence of α1/ α1, β, γ-globulin as well as albumin. The presence of such plasma proteins is due to the separation of rabbit serum as a result of the IgG purification step.

Utilising the Bradford protein assay to determine protein concentrations of purified IgG and rabbit serum

To determine the protein concentrations of the samples the mean absorbance of bovine serum albumin (BSA) were plotted against its protein concentrations GRAPH. Thereafter, using the known purified IgG and serum dilution absorbencies their undiluted protein concentrations were calculated .

A linear correlation was found between BSA protein concentration and the absorbance GRAPH. Furthermore, a decrease in the protein concentrations was observed as the dilution factor increased .this data from the graph allowed for the neat-undiluted protein concentration to be calculated with the multiplication of graph values by the dilution factor . Moreover, the mean protein concentration was also determined by dividing the sum of the undiluted protein concentration by the number of dilutions carried out.consequently, the mean protein concentration was 1 mg/ml for BSA, 44.42 mg/ml for the serum and 0.51 mg/ml for the purified IgG.

Legends

Statistical analysis of group data RED/how to do sd/cv

Standard deviation: √∑ (x-x)2/n-1

Coefficient of variation = Standard deviation / Mean

The application of standard deviation and coefficient of variation on the group results showed that the variation of the data from the mean is 14.805 for the serum and 0.221for the IgG protein concentration. Moreover, this statistical analysis of the data omitted any anomalies which are highlighted in red ().The significant variation of the values from the mean for serum concentration, illustrated by , is caused by the noticeable differences of the values obtained between groups. An example of this can be demonstrated with the results of H.Ibbish and Z.Abbas group which had a serum concentration of 13.87 mg/ml compared to S.Sharif and S.Mahmood who had 80.80 mg/ml. Coefficient of variation gives further support to the variance of the data from the mean as 0.333 was calculated for serum and 0.370 for IgG protein concentration.

Identification of IgG on nitrocellulose blots of samples separated by agarose gel electrophoresis

Following the transfer of proteins to the nitrocellulose paper 0.5% Ponceau S solution in 1% acetic acid was applied resulting in resulting in a pink coloured residue at the sites where proteins were present. Removal of Ponceau S solution followed by the addition of diluted peroxidise-conjugated sheep anti-rabbit IgG followed by 4-chloronaphthol, was used to visualise IgG. The subsequent oxidation of 4-chloronaphthol by peroxidise resulted in a blue insoluable product, which identifying the site where IgG was present . A diagram of this process can seen .

Pon s & kauser pic here

The application of 4-chloronaphthol to the nitrocellulose paper resulted in two separate distinct blue stains .

The well to the right has formed significantly more blue product indicating the presence of purified IgG. The adjacent well has weaker staining due to less IgG present, indicating that the well contains rabbit serum.

Discussion

Principles of BSA, Electro,western>PDFs

Discuss above results>electro 15 min>affects sep>igg>

The aims of these experiments were to determine the concentration of IgG, both in its purified form and in the rabbit serum. This was achieved by utilising a range of techniques including the Bradford protein assay, electrophoresis and western blotting.

To obtain purified IgG from the rabbit serum octanoic acid was used which precipitates out the non-IgG proteins from the sample wirh subsequent centrifugation and extraction of the purified IgG. This simple method can purify more than 80% of IgG from the rabbit serumREF.

Using the Bradford protein assay

The Bradford protein assay was used to determine the protein concentration of the samples. This assay is based upon the shift in the absorbance due to a change in the Coomassie dyes normal red colour to Coomassie brilliant blue, when stably bound to proteins. Therefore, the amount of stable Coomassie blue formed is proportional to the protein concentration, which can be measured by the spectrophotometer at the wavelength of 595nmREF. However, due to a low linear range, as well as the limited absorbance range of the spectrophotometer, solutions with high protein conectrations must be diluted, as was the case for the purified IgG and the serum. The protein concentrations were calculated at 44.42 mg/ml and 0.51 mg/ml for the rabbit serum and the purified IgG respectively, resulting in 1.15% of the serum protein being recovered in the IgG prep.

The statistical analysis of group results was carried out by calculating the standard deviation and the coefficient of variation. The standard deviation determines the variation from the mean value. The group rabbit serum mean was 44.49 mg/ml and a standard deviation of 14.805, while the purified IgG mean was 0.60 mg/ml with a value of 0.221 for the standard deviation. Moreover, these statistics were calculated with the anomalies omitted , consequently, resulting in a more accurate analysis. In any case, the large value for the serum standard deviations reflects the dispersion of the data from the mean indicating that there are significant differences between the results of each group. The purified IgG standard deviation was a lower value, showing a smaller dispersion of the results about the mean. However, the coefficient of variation, which looks at the ratio of the standard deviation to the mean, was noticeably high for both the serum (0.333) and IgG (0.370). A high coefficient of variation value demonstrates the inconsistency of the values within the group.

Electrophoresis

Agarose gel electrophoresis was used to allow the proteins present within the samples to migrate and separate under an electric field. The structure of the agarose gel polysaccharide is such that pores are present, by altering the agarose concentration, through which molecules may pass REF. The application of an electric field allows for a negatively charged sample, for example, to migrate from the cathode to the anode. However, due to the structure of the agarose matrix, the smaller molecules migrate further than the larger molecules.REF Consequently, prior to loading the serum onto the agarose gel it was diluted in order to allow the proteins effectively pass through the small pores and migrate along the gel and form distinct bands according to the molecular size of the proteins, which would have been difficult to achieve if the neat serum sample was used. Alternatively SDS-polyacrylamide gel electrophoresis, based on the same principles as agarose gel electrophoresis, could have also been used to separate the proteins in the samples and subsequently processed further by the western blotting.

Following electrophoresis, various distinct protein bands could be visualised including for purified IgG, β, α1 and α2-globulins as well as albumin. In general the movement of the proteins was from the cathode to the anode as a result of the these proteins having a net negative charge. In addition to the movement of β, α1 and α2-globulins, the greatest migration was by albumin. This shows that albumin was not only negatively charged but had the smallest molecular size of the proteins and was present in the most significant quantaties in the rabbit serum, demonstrated by the greater staining . Purified IgG as well as γ-globulin remined at the site of origin. This was due to the high molecular size of IgG as well as the fact that IgG and γ-globulin had no net electrical charge as they were at their isoelectrical point, the pH at which the proteins net charge is neutral.

The nitrocellulose paper was stained with Ponceau S solution to detect the presence and therefore the successful transfer of proteins to the paper. The protein bands were clearly visualised demonstrating the a successful blot . In order to detect the presence of IgG peroxidise conjugated sheep anti-rabbit IgG, which is specific for IgG, was added to the blot. Subsequent addition of the substrate 4-chloronaphthol that reacts with the peroxidase on the sheep anti-rabbit IgG, resulted in a blue coloured product. Therefore, the formation of this coloured produt not only demonstrates the fact that IgG is present on the nitrocellulose, but it is pure IgG due to the clear strong staining on the nitrocellulose . Contamination is a possibility, such as from other similar immunoglobulin's such as IgA and IgE etc, but if it occurs will be minimal. Furthermore, our findings showed to evidence of such contamination.

Stronger staining was established by the peroxidase-antibody than Coomasssie blue as Coomassie dye binds to all proteins producing a more diffused colour. The peroxidase anti-IgG complex, however, is more specific and so binds only to rabbit IgG giving an intense concentrated localised staining.

In conclusion,

To do

Discussion; errors-s.d-electro/principles/

s.d

s with legends

graph

read thru and improve/reword CLASS

structure!

REFs

handbook

Bradford, M. (1976). Anal. Biochem.72, 248-254.

1. Bradford, M., "A Rapid And Sensitive Method For The Quantitation Of Microgram Quantities Of Protein

Utilizing The Principle Of Protein-Dye Binding," Anal. Biochem., 72, 248 (1976).

Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY

A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid

Citation

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