Calculation of molecular weight of the unknown protein sample is a 5 step process:

1. Resolve the protein sample on the SDS-PAGE along with the molercular weight markers.

2. Calculate the relative mobility (Rf) using the following formula:

(13.6)

3. Plot log molecular mass (y-axis) versus relative mobility (x-axis) of the standards.

4. Perform a linear regression using a calculator or using regression software such as Microsoft Excel.

5. Use the linear regression equation (y=mx+c) to estimate the mass of the unknown protein.
Log Molecular Weight=(slope)(mobility of the unknown)+Yintercept

Troubleshooting : The pattern of the protein bands on polyacrylamide gel depend on several factors. As a result several types of defects are observed. Now we will discuss few of these defects and responsible factors.

1. Smiling : Uneven heating of the gel causes differential migration of proteins, with outer lanes moving slower than the middle lanes. Rapid heat transfer eliminates this defect and be achived by filling the lower tank with the buffer until sample height.

2. Diffuse Protein Bands: Diffused pattern of the protein band appears on the PAGE. Diffused protein bands pattern can be corrected by increasing running current by 25-50%, higher concentration of acrylamide.

3. Vertical Streaking: Vertical streaking of the protein bands appers due to over-loading of the protein sample. It can be corrected by either reducing amount of the protein sample or running the gel at lower current.

4. Protein Runs faster than expected: In few cases migration of protein is not proportional to the molecular weight, it is either more or less on gel than the expected place. It is due to the unusual very high proportion of basic or acidic amino acids.

5. Double Bands: Appearance of double bands is due to partial oxidation or degradation of the protein sample. Addition of more amount of reducing reagent or preparing a fresh sample will reduce these artifacts.

6. Distorted Protein Bands: Appearance of distorted or uneven protein band is due to stacking gel polymerization. It can be corrected by increasing amount of ammonium persulfate and TEMED or deaerating the stacking gel.

7. Lateral Spreading: if protein bands appears laterally spreading, it can be avoided by reducing the time between loading of the sample and running.