Western Blot (Denaturing Condition) - 1. Sample Preparation

Western blot is used to detect proteins in the sample. In this technique, proteins are separated by their molecular weight during gel electrophoresis. The proteins in the gel are then transferred to a membrane. Then the membrane is incubated in the antibody solution to detect proteins. Most of western blot is processed under denaturing condition. However, in certain circumstances, western blot should be processed under non-denaturing conditions. Uploading Western Blotting under Non-denaturing Condition will be followed by uploading Western blotting under denaturing Condition.

Procedure Sample preparation

1. Add 50 ug of protein into an ep tube. Note1. The amount of protein can be adjusted. Note2. The concentration of protein should be over 5 mg/ml to get more reliable concentration.

2. Add DW to be a final volume of 10 ul. Note. The final volume can be less than 10 ul. But do not exceed the volume of SDS-PAGE gel well capacity.

3. Add 10 ul of 2X SDS sample buffer containing β-mercaptoethanol to be a final concentration of 1X. Note. Before using 2X SDS sample buffer, add 50 ul of β-mercaptoethanol (BME) into 950 ul of 2X SDS sample buffer. BME reduces the disulfide bonds in protein structure causing unfolding of the protein. See β-mercaptoethanol.

4. Brief vortex and pulse spin down.

5. Boil the sample at 95°C in a heating block for 5-10min to denature the protein. Note1. Place a spare block. Boiling the samples in ep-tubes may make the cap pop open. Note2. See Why We Boil the Samples.

6. (Option)Incubate for 20 min on ice.

7. Centrifuge at 12,000g for 1 min at 4°C. Note. Samples can be stored at -20°C for up to 1 month. Boiled sample doesn't need to be re-boiled after thawing. But avoid freeze-thaw cycles.

8. Load the samples on the gel. Note. Don’t forget to load protein ladders.

Sample Buffer for Western Blotting under denaturing condition 2X SDS sample buffer (= 2X Laemmli buffer), store at RT.

Role of compounds

• Glycerol: Adds density to samples so that samples can be loaded and maintained at the bottom of the well.

• Tris-HCl: Adjusts or stabilizes the pH of the solution.

• SDS: Denatures proteins and coating them negatively charged so that they can be moved to one direction during electrophoresis.

• BPB (bromophenol blue): Used as a tracking dye. Its color turns from blue to yellow if you used acidic lysis buffer but the color changes won’t affect the results.

β-mercaptoethanol (BME) As BME is very volatile, the concentration in solutions decreases with time. Thus, it has to be added into sample buffer just before sample preparation. Ideally, once BME was added to 2X SDS sample buffer, it should be kept on ice and shouldn’t be re-used. However, practically, if make 1 ml of 2X SDS sample buffer containing BME, aliquot and store at -20°C. Then, it can be used for up to 1 year.

Or DTT can be used instead of BME. For DTT, use 5-10 mM while a concentration of 5% is needed for BME.

Why We Boil the Samples

Boiling the samples is required to ensure that samples are truly denatured. Once you boiled the sample, it can be stored at -20°C for up to 1 month. Denaturing proteins is very important in SDS-PAGE otherwise the protein would be detected at the less size than its original molecular weight since SDS couldn't coated proteins negatively charged enough.

But boiling sample is not required every occasions. If you want to detect membrane proteins, boiling the samples causes aggregation. Thus, the sample should be incubated at RT for 30 min for membrane protein detection.