双向[凝胶]电泳（英语：Two-dimensional [gel] electrophoresis）是一种等电聚焦电泳与SDS-PAGE相结合，分辨率更高的蛋白质电泳检测技术。双向电泳后的凝胶经染色蛋白呈现二维分布图，水平方向反映出蛋白在等电点上的差异，而垂直方向反映出它们在分子量上的差别。所以双向电泳可以将分子量相同而等电点不同的蛋白质以及等电点相同而分子量不同的蛋白质分开。双向电泳是快速成长的蛋白质组学技术中最流行最通用的蛋白质分离方法。目前2D-PAGE能够在同一块凝胶上同步检测和定量数千个蛋白质。

Two-dimensional gel electrophoresis, (2-DE or 2D electrophoresis), is a form of gel electrophoresis commonly used to isolate the proteins in a sample for further characterization by mass spectroscopy. Mixtures of proteins are separated in the 1st dimension by their charge (isoelectric focusing (IEF)). Then in the 2nd dimension, proteins are separated by their size (molecular weight). This video shows the part 2- protein Resuspension&Quantitation.

配制水化液

水化液需当天新鲜配制(可配成储液分装-20 度保存，但不可反复冻融)。尿素溶液加热温度不能超过 37°C，否则蛋白会发生氨甲酰化。 

Bradford蛋白定量

The Bradford protein assay is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is subjective, i.e., dependent on the amino acid composition of the measured protein. The Bradford protein assay was developed by Marion M. Bradford.

Principle

The Bradford assay, a colorimetric protein assay, is based on an absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer form to bind to the protein being assayed. During the formation of this complex, two types of bond interaction take place: the red form of Coomassie dye first donates its free electron to the ionizable groups on the protein, which causes a disruption of the protein's native state, consequently exposing its hydrophobic pockets. These pockets on the protein's tertiary structure bind non-covalently to the non-polar region of the dye via van der Waals forces, positioning the positive amine groups in proximity with the negative charge of the dye. The bond is further strengthened by the ionic interaction between the two. The binding of the protein stabilizes the blue form of the Coomassie dye; thus the amount of the complex present in solution is a measure for the protein concentration, and can be estimated by use of an absorbance reading.
The (bound) form of the dye has an absorption spectrum maximum historically held to be at 595 nm. The cationic (unbound) forms are green or red. The binding of the dye to the protein stabilizes the blue anionic form. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample.
Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. An exception of note is elevated concentrations of detergent. Sodium dodecyl sulfate (SDS), a common detergent, may be found in protein extracts because it is used to lyse cells by disrupting the membrane lipid bilayer. While other detergents interfere with the assay at high concentration, the interference caused by SDS is of two different modes, and each occurs at a different concentration. When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution. When SDS concentrations are above CMC, the detergent associates strongly with the green form of the Coomassie dye, causing the equilibrium to shift, thereby producing more of the blue form. This causes an increase in the absorbance at 595 nm independent of protein presence.
Other interference may come from the buffer used when preparing the protein sample. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. This will not be a problem if a low concentration of protein (subsequently the buffer) is used.

Disadvantages

The Bradford assay is linear over a short range, typically from 0 µg/ml to 2000 µg/ml, often making dilutions of a sample necessary before analysis.
It is also inhibited by the presence of detergents.
Much of the non-linearity stems from the equilibrium between two different forms of the dye which is perturbed by adding the protein. The Bradford assay linearizes by measuring the ratio of the absorbances, 595 over 450 nm. This modified Bradford assay is approximately 10 times more sensitive than the conventional one. (Zor & Selinger, 1995)

Sample Bradford procedure

Materials

• Lyophilized bovine plasma gamma globulin or bovine serum albumin (BSA)
• Coomassie Brilliant Blue 1
• 0.15 M NaCl
• Spectrophotometer and tubes
• Micropipettes

Procedure (Standard Assay, 20-150 µg protein; 200-1500 µg/ml)

1. Prepare a series of protein standards using BSA diluted with 0.15 M NaCl to final concentrations of 0 (blank = NaCl only), 250, 500, 750 and 1500 µg BSA/mL. Also prepare serial dilutions of the unknown sample to be measured.
2. Add 100 µL of each of the above to a separate test tube (or spectrophotometer tube if using a Spec 20).
3. Add 5.0 µL of Coomassie Blue to each tube and mix by vortex, or inversion.
4. Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube which contains 0 BSA.
5. Wait 5 minutes and read each of the standards and each of the samples at 595 nm wavelength.
6. Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.

Procedure (Micro Assay, 1-10 µg protein/mL)

1. Prepare standard concentrations of BSA of 1, 5, 7.5 and 10 µg/mL. Prepare a blank of NaCl only. Prepare a series of sample dilutions.
2. Add 100 µL of each of the above to separate tubes (use microcentrifuge tubes) and add 1.0 mL of Coomassie Blue to each tube.
3. Turn on and adjust a spectrophotometer to a wavelength of 595 nm, and blank the spectrophotometer using 1.5 mL cuvettes.
4. Wait 2 minutes and read the absorbance of each standard and sample at 595 nm.
5. Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.