![]() ![]() If you are having problems getting your antibody to work, make sure that you have checked the obvious: Consider adding a small amount of SDS to the transfer buffer and increasing the transfer time if this is your problem.Īntibodies vary tremendously some are fool proof while others are tricky as heck. Some large proteins may not transfer at all. Large proteins have the opposite problem. If you are working with a small protein, test different transfer times and use two membranes during the transfer. Small proteins move faster than large proteins during transfer and can actually transfer through the membrane. Make sure yours is too, by using extra sponges if needed using appropriately thick filter paper (too thick can cause issues) and as always insure that all of your sandwich components are clean, hydrated and free from bubbles. A common source of transfer problems is the tightness of the sandwich. To avoid this problem, simply run your transfer at a lower voltage for a longer time. Transferring the gel too quickly and hot.Luckily there ARE reasons behind these bad transfers: These problem are usually witnessed after you transfer when you stain your membrane and gel with Ponceau S or Coomassie for protein detection. Transfers with swirls, mystery protein splotches, loss of protein, or a general variability in transfer efficiency are common Western blot problems. It is never a bad idea to look for a few bubbles (a sure sign that SOMETHING is happening).Įven if you correctly set up your transfer, and double-checked the things above, a transfer can still go wrong. Stay a few minutes to make sure that your voltage and amps are okay. Do not just hit “transfer” and walk away. If there is a timer on the gel box make sure that it doesn’t turn your transfer off too early! Oh, the lamentations I have heard when this is done improperly! Did you plug it in right? Double check that the electrodes are plugged into the correct positive/negative outlet.Do you know where your proteins are? Double (or triple check) that the gel is in the correct place relative to the membrane, and that you know the orientation of your lanes.Is everything in the sandwich properly hydrated? Were there no bubbles, including on the hydrophobic membrane?.Did you handle the membrane properly? That is, did you handle it with gloves, and ensure that there are no pressed marks if using laser capture?.To minimize your number of stupid mistakes, always ask yourself the following questions before you start ANY Western blot transfer (no matter how routine): In the laboratory it is common to be multitasking, overworked and distracted-all things that lead to stupid mistakes. While we like to think that we are above stupid mistakes, we are not. It is faster to make a new gel than to do a new sample preparation AND a new gel. If you see any of these problems, chuck your gel and start over. Before you use your gel, gently tilt it to ensure that it is completely set up. If your gel can be seen shrinking from the edge, it is drying out. These imperfections will impair protein migration. Properly made wells should be uniform and rinsed. To avoid losing your samples to a bad gel, always examine your gel closely before use. A Bad GelĪfter hours of performing experiments and preparing samples, you better make sure your samples are loaded onto a pristine gel! However, even if you do everything right-you followed the recipes carefully, minded you pH, used fresh reagents, meticulously cleaned your plates-things can still go wrong with your gel. An optimize protocol will maximize your protein-of-interest’s solubility and stability, which will allow you to better visualize your protein and reliably interpret your Western blot data. Proper sample preparation is an empirical process involving the comparison of detergent extraction versus whole cell lysate, salt concentrations, buffer types, inhibitors (to prevent degradation and loss of post-translational events), reducing agents and heating times. While labs often have their own sample preparation methods, consider optimizing your own process. Bad protein samples due to improper or sub-optimal protein preparation can result in poor Western blots. Proper protein extraction and sample preparation is critical. While some of these mistakes are perplexing, others are just plain dumb but none of them have to happen to you. Sadly this is usually due to mistakes on the experimenter’s part. As bitter experience has likely taught you, not all Western blots are pretty. ![]()
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