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Using SDS-PAGE and Scanning Lar Densitometry to Measure Yield and Degradation of Proteins
经典国学诵读
Aaron P. Miles and Allan Saul
1. Introduction
A variety of techniques are available for the quantification of proteins, their degradation products and other impurities. Densitometry is particularly uful due to its nsitivity, accuracy and versatility, and it can be applied to proteins in gels or on membranes and ud with numerous detection methods. In addi-tion to being accurate, nsitive and reproducible, the technique is cost-effective, simple, and does not require a high degree of specialized training, yet provides technical advantages over other available tools. For example, Vuletich and Osawa showed that densitometry following SDS-PAGE and electroblotting was 20-fold more nsitive than HPLC at detecting oxidatively modified myoglobin (1). In addition, Morçöl and Subramanian reported the development of a nsi-tive Ponau-S-stained dot blot/densitometric protein assay that was less subject to interference by pH, detergent and other reagents than the commonly ud Bradford protein assay (2). More recently, Zhu et al. and Miles et al. reported a quantitative densitometric method that measures host cell derived impurity levels on immun
oblots of recombinant proteins expresd in E. coli (3, 4). This method was shown to be at least 20 times more nsitive than a commercially available ELISA kit designed to measure host cell impurity levels (3). Other groups have reported on the utility of gel or fluorograph densitometry, using multiple detection methods, in carbohydrate analysis (5), taxonomic and forensic applications (6), as a clinical diagnostic tool in Balkan nephropathy (7) and in the analysis of high density lipoproteins (8), as a means of quantifying relative levels of excretory-cretory polypeptides synthesized in vitro by Schistosoma mansoni daughter sporocysts (9), and for analyzing adsorption of proteins and an oligodeoxynucle-otide in Alhydrogel®-bad malaria vaccine candidates (10).
From: The Protein Protocols Handbook, Third Edition
Edited by: J.M. Walker © Humana Press, a Part of Springer Science + Business Media, LLC 2009
487
488 Miles and Saul As a typical example we describe here the analysis of recombinant proteins by SDS-PAGE and scanning lar densitometry. This approach offers advan-tages over other techniques for two main reasons: 1) proteolytic degradation is common among the proteins, and this method is able to accurately quantify it in both minute and large amounts, and 2) the common co
nstituents of fermenta-tion and refolding tanks and purification process do not cau interference, so it can be ud at any stage throughout production and purification. Analysis of a recombinant malarial protein at two different stages of purity will be ud to illustrate this method.
2. Materials
1. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) equip-
ment.
2. Coomassie Blue staining solution: 40% methanol, 10% glacial acetic acid, two
PhastGel Blue R tablets (Amersham Biosciences, Piscataway, NJ) per liter.
3. Coomassie Blue destaining solution: 5% methanol, 10% glacial acetic acid.
4. 0.45-micron filtration unit.
5. Concentrated, diafiltered test protein (Pvs25H) fermentation culture supernatant.
6. Purified test protein (Pvs25H) solution.
7. Personal Densitometer SI with ImageQuant software (Molecular Dynamics Inc.,
Sunnyvale, CA, [11]).
3. Methods
The methods described below outline (1) the running of SDS-PAGE gels fol-lowed by visualization with Coomassie Blue, and (2) scanning and analyzing the gels using lar densitometry and ImageQuant software.
3.1. SDS-PAGE
1. Load the gel with samples as desired, and run according to the manufacturer’s
instructions.
2. Filter the staining solution using a 0.45 μm filtration unit (e Note 1).
3. Add staining solution to the gel, bring to a boil in a microwave oven, and agitate疫情下的我们
for 30 min (e Notes 2 and 3).算天数
4. Pour off staining solution, add destaining solution, bring to a boil, and agitate for
大海作文15 min (e Note 4).连通器的原理
5. Change destaining solution, bring to a boil again, and continue agitation for 15
more min.
6. Change destaining solution once more, bring to a boil, and continue destaining
until gel background is clear or almost clear, approximately 10 min. Take care not to overly destain the gel as minor protein bands can disappear (e Note 5).
7. Place the gel in water until scanned. Fig. 1 shows a nonreduced 4–20% Tris-glycine
SDS-PAGE gel that contains standards of purified Pvs25H (a recombinant form of
给背影唯美简短一句话Using SDS-PAGE and Scanning Lar Denistometry 489the 25 kDa ookinete suface protein from the malaria parasite Plasmodium vivax ,
[12]) and a concentrated, diafiltered fermentation culture supernatant of the same protein. Fig. 2 sho
ws a reduced 15% acrylamide gel loaded with the same purified protein that was subjected to 37°C storage for up to 14 days to monitor stability (e Note 6). This protein is heavily disulfide bonded and must be reduced in order to visualize the degradation occurring over time. The gels will rve as examples of two applications of scanning lar densitometry: (1) monitoring low levels of degradation in a highly purified protein, and (2) quantifying yields of a protein
recovered in a fermentation broth not yet subject to purification.
Fig. 1. SDS-PAGE of Pvs25H Fermentation Culture Supernatant with Purified Pvs25H Standards. Nonreduced 4–20% Tris-glycine SDS-PAGE Coomassie Blue-stained gel showing purified Pvs25H standards and concentrated, diafiltered fermentation culture supernatant. Purified Pvs25H was loaded in the indicated amounts for comparison to Pvs25H in the fermentation supernatant.酱油跟生抽的区别
490 Miles and Saul
Fig. 2. SDS-PAGE of Purified Pvs25H Stored at 37°C. Reduced 15% SDS-PAGE Coomassie Blue-stained gel showing samples taken at the indicated time points during storage at 37°C. All protein loads are 2 μg, parated by blank lanes. *A minor band accumulating with time who quence corresponds to cleavage at a single site within Pvs25H. **A minor band (Pvs25H-derived) prent in final purified Pvs25H, not increasing upon storage. This type of gel was chon to better resolve Pvs25H, which migrates diffuly in a gradient gel (e Fig. 1).
Fig. 3. Gel from Fig. 2 with Lines Drawn in ImageQuant for Analysis. Shown is the same gel as in Fig. 2, with lines drawn down two lanes using ImageQuant. The lines define the length and width of the areas being analyzed. Lengths and widths are adjusted to encompass all protein in each lane. The lines give ri to the line graphs shown in Fig. 5.
Using SDS-PAGE and Scanning Lar Denistometry 491
3.2. Lar Densitometry
1. Scan the gel according to manufacturer’s instructions, ensuring that the lanes of
古筝教师the gel run straight from the top to the bottom of the scanning bed.
2. Draw an encompassing line for analysis down each lane of interest and widen the
line appropriately. Include in the analyzed area of each lane enough length from top to bottom and enough width to envelope all visible protein bands. Figs. 3and
4 illustrate this, using the same gels as shown in Figs. 2 and 1, respectively.
3. Create a line graph for each line drawn. Examples are shown in Fig. 5, which shows
reprentations (in optical density units) of purified Pvs25H stored for one (A) and
14 (B) days at 37°C, and Fig. 6, which shows Pvs25H in concentrated, diafiltered
Fig. 4. Gel from Fig. 1 with Lines Drawn in ImageQuant for Analysis. Shown is the same gel as in Fig. 1, with lines drawn down each lane containing protein. The line drawn down the fermentation supernatant lane gives ri to the line graph shown in Fig. 6.
