| 1) |
The primary and secondary antibodies used, the catalog number, company purchased from, and lot number if it is a polyclonal antibody. |
Possibly by recognizing alternatively spliced variants, PTM configurations etc. In a few cases antibodies can recognize non-specific bands around the molecular weight of the target protein. Different antibodies to the same protein can give different results. Currently more polyclonal antibodies are used for Western blotting but vary from lot to lot due to different animals, improper storage, and different bleeds from individual animals. |
| 2) |
Molecular mass of band of interest should be shown on the blot. |
Several antibodies recognize bands which are not the proper molecular masses of the target protein. In fact some antibodies that are known to recognize only a different molecular weight protein are sold by some companies. |
| 3) |
The amount of total protein loaded onto the gel and the method used to determine the protein concentration should be stated. |
Use of too much protein results in inaccurate quantification. |
| 4) |
Type, amount and extent of use of blocking reagent. |
The type and amount of blocking reagents can significantly affect the number of non-specific bands detected by an antibody. |
| 5) |
Washing solution used, how often and for how long. |
The buffer used and amount of washing affects the background membrane staining and sometimes the number of non-specific bands detected by an antibody. |
| 6) |
Amount and incubation time of primary antibody. Primary antibody buffer. |
The primary antibody amount and buffer it is diluted in are critical for good Western blots. |
| 7) |
Amount and time of incubation of secondary antibodies. Secondary antibody buffer. |
The secondary antibody amount and buffer used is important for good Western blots. |
| 8) |
How the image was collected. |
If X-ray film was used then the source of X-ray film is needed. X-ray film has lower linearity than commercial imagers. |
| 9) |
The reagent used for detection and how long the membrane was incubated in the reagent (including company that manufactured the reagent). |
Different ECL reagents typically have distinct properties. Different fluorescent secondary antibodies also sometimes have dissimilar properties. |
| 10) |
The software (including company that created the program and version number) used for data analysis. Basic information about how the quantification was carried out. |
If quantification is incorrectly done then no matter how well the Western blot was carried out the data from the Western blot will be inaccurate. |
