Secondly, your loading control protein should have a different molecular weight than the protein of interest, this ensure the bands are distinct and expression levels of your protein are quantifiable. For this reason, ‘housekeeping’ genes are frequently chosen as loading controls. What makes a good loading control you ask? You should look for proteins that exhibit high level, constitutive expression in the cell type you are examining, as this means it will have constant high expression levels. They can be used to quantify the protein amounts in each lane and normalise the expression levels of your protein of interest to the loading control Once you have established there are not large differences in protein levels, loading controls can then be used to normalise small variations in levels.Loading controls can show if this has occurred and allows for this variation to be accounted for. This can result in more intense stains at the edge than in the middle of the gel. This helps to protect against what is known as the “edge effect”, that is when large numbers of lanes are being used, proteins in the outer lanes transfer to the membrane in a position close to the frame.For example, they can be used to check that there has been even transfer from the gel to the membrane across the whole gel.They allow you to check that protein loading is the same across the gel, the expression level of the loading control should not vary between the different sample lanes. Loading controls are essential for publication worthy Western Blots.Why do I need a loading control for Western blotting? We provide a guide to some of the most commonly used types of loading control antibodies. Antibodies against several different targets are commonly used and it’s not always obvious which you should choose. # AT0098-100ul, Engibody) in conjunction with your chemiluminescence reagents and instrumentation.Loading control antibodies are some of the most utilised antibodies in research. # AT0097-100ul, Engibody) or goat anti-mouse IgG conjugated HRP (Cat. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20.ĭetect the primary antibody band using an appropriate secondary antibody, such as goat anti-rabbit IgG conjugated HRP (Cat. Incubate the blocked blot with primary antibody at a 1:1000 starting dilution in Tris buffered saline supplemented with 1% Ig-free BSA and 0.1% Tween-20 overnight at 4☌ or for two hours at room temperature. Transfer the proteins at 140 mA for 6☌ 0-90 minutes at room temperature.įollowing the transfer, rinse the membrane with Tris buffered saline for 2 minutes.īlock the membrane with blocking buffer (formulation provided below) overnight at 4☌ or for one hour at room temperature. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes.Īssemble the gel and membrane into the sandwich apparatus. Alternatively, nitrocellulose may be used. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. In preparation for the Western transfer, cut a piece of PVDF slightly larger than the gel.
Load 10-30 µg of the cell lysate into the wells of an appropriate single percentage or gradient mini-gel and resolve the proteins by SDS-PAGE. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol ) and boil the mixture for 90 seconds at 100☌. Polypropylene tubes are recommended for storing cell lysates. Alternatively, lysates may be ultra-centrifuged at 100,000 x g for 30 minutes for greater clarification.Ĭarefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. This cell lysis buffer formulation is available as a separate product which requires supplementation with protease inhibitors immediately prior to use (Cat. This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. Lyse approximately 107 cells in 0.5 mL of ice-cold Cell Lysis Buffer (formulation provided below).