western blot band intensity quantification

#WESTERN BLOT BAND INTENSITY QUANTIFICATION HOW TO#
#WESTERN BLOT BAND INTENSITY QUANTIFICATION SOFTWARE#
#WESTERN BLOT BAND INTENSITY QUANTIFICATION CODE#

To reduce proteins completely, boil the sample in SDS-PAGE buffer containing a denaturing agent such as dithiothreitol or β-mercaptoethanol for 10 minutes rather then the usual 5 minutes. Unresolved protein multimers can result in molecular weight bands that are higher than the predicted molecular weight of the target protein. Higher molecular weight bands alone can also be due to technical artifacts. Keep samples on ice during preparation and during all manipulations to limit protease activity.Īliquot samples into multiple tubes and freeze/thaw each tube only 1-2 times to limit degradation induced by temperature changes. To decrease degradation of proteins by cellular proteases, include protease inhibitors in the lysis solution used for sample preparation. Multiple lower molecular weight bands usually arise from poor handling of the lysates/samples prior to analysis and can be indicative of degradation of the sample. Commercial kits are available for affinity purification of antibodies. Alternatively, polyclonal antisera can be affinity purified using immobilized Protein A, G or L to purify IgG molecules or immobilized antigen can be used to purify antigen-specific antibodies. When possible, purchase affinity purified antibodies. Polyclonal sera or unpurified antibodies can also contain less abundant antibodies that bind to abundant, common cellular proteins. To decrease non-specific binding to proteins of greater abundance increase the number and duration of washes. If the protein of interest is not very abundant, than it may be necessary to load large amounts of protein to detect the protein. To determine the appropriate amount of lysate to load on the gel, load decreasing dilutions of lysate on the gel. If too much lysate is loaded onto a gel, antibodies can bind non-specifically to proteins of excessive abundance, resulting in multiple bands. If multiple bands are still observed, then the secondary antibody is responsible for the artifacts. To determine quickly whether the primary antibody is responsible for producing multiple bands on the blot, perform the entire Western blot procedure but omit the primary antibody. Both antibodies can be titrated simultaneously using a dot blot in a checkerboard like fashion. To determine if high antibody concentrations are resulting in multiple bands, titrate the antibodies. Non-specific binding can occur with any protein, thus bands of higher and lower molecular weight may be observed. If the concentration of either the primary or secondary antibody is too high, the antibody can bind non-specifically to proteins other than the protein of interest. The types of bands that are observed can help determine the cause of the artifact.Ĭoncentration of Primary or Secondary Antibody too High Technical artifacts can cause the appearance of bands that have higher or lower molecular weights than the actual protein. Multiple Bands Caused by Technical Artifacts

#WESTERN BLOT BAND INTENSITY QUANTIFICATION HOW TO#

This guide describes various causes of multiple bands due to technical artifacts and how to determine if multiple bands represent true variants of the protein of interest. When dealing with multiple bands on Western blots, it is important to determine whether they are due to technical artifacts or whether they represent true variants of the protein of interest. However, analysis of the protein can be difficult if multiple bands appear on the blot. ipynb notebooks for details/example usage.The Western blot assay provides valuable information about a protein including abundance, the apparent molecular mass, post-translational modifications and splice variants.

#WESTERN BLOT BAND INTENSITY QUANTIFICATION CODE#

The pipeline is functional and accurate but slow old code is preserved to reflect analysis in. Note: updates needed for SDS-PAGE gel analysis (spotting assay approach is much faster/better implemented).

This data is plotted and can be quantified for band intensities. Data for each slice is baselined given a user-specified baselining region. Then splits image into user-specified lanes/boxes, and the average intensity of every pixel per lane/band/blot/spot is calculated.įor gels, intensities have the option to be gaussian-weighted so that the middle of the gel band contributes more to the average than the outer edges, as other lanes in gels sometimes bleed into the lane of interest. Takes an input image which gets cropped by the user and converts to a grayscale numpy array using the Python Image Library (PIL). Currently implemented for protein and DNA gels/blots and yeast spotting assays.

#WESTERN BLOT BAND INTENSITY QUANTIFICATION SOFTWARE#

Simple python-based quantification software for simple biological images.

Western blot band intensity quantification