western blot ladder transferred but no proteins

Again, this is a “post-hoc” check for transfer efficiency: once you’ve stained the gel, you won’t be able to set it up for a second transfer. This is a quick and easy procedure that will give you a good idea of how much protein is still in the gel: if the gel is mostly clear, than your transfer was successful, but if most of your gel shows blue bands, then your transfer needs to run longer. After transfer, stain the SDS-PAGE gel with Coommassie blueĪnother simple way to monitor the efficiency of protein transfer is to stain the gel with Coommassie after your transfer is complete. Remember, if you’ve opened your transfer cassette to “peek” at the ladder, this will introduce air bubbles to the transfer stack – so you probably want to use this technique to optimize transfer times when you’re not running important samples. If you use a ladder where each band is stained a different color, this can help you keep track of the transfer efficiency of differently sized proteins. Instead, you are aiming for a point at which most of the larger proteins have migrated to the membrane, and most of the smaller proteins are still on the membrane but not through it. Keep in mind that proteins of different sizes will migrate at different speeds: there is no magic moment at which all the proteins jump from the gel to the membrane.

The rest of the proteins on your gel will of course be invisible, but you can easily see if the brightly colored ladder bands are still in the gel, or have transferred completely to the membrane. Use a pre-stained protein ladder to track transfer of proteins from a gel to a membrane. So how do you know when your transfer is complete? Here are a few good controls you can use to monitor transfer efficiency: Look for complete transfer of the molecular weight ladder Instead, you will need to optimize the transfer time and conditions for yourself, based on the equipment you use and the proteins you are working with. Transfer times are empirical and based on your own particular samples, which means that there is no easy way to determine how long you need to run the transfer in order to get complete transfer of all your proteins.

Now it’s time for the all-important transfer step, that tricky point that will determine the quality of your Western blot. So, you’ve done your experiment, prepped your samples, and run your SDS-PAGE gel.

Western blot ladder transferred but no proteins