Independent experiments and controls. AKA, literally the basis of experimental science.
Let's say you want to know where a protein localizes in a cell, of a given tissue, in both mutant and wild-type organisms.
* Immunolocalization. You develop antibodies to the protein of interest, fix and mount tissue, perfuse it with the antibody, and use a secondary antibody to make it detectable.
* Fluorescent tagging. You make a construct with your protein fused to a fluorescent protein. Usually this involves trying a few different tagging strategies until you find one that expresses well. Then you can make a stable transgenic, or try a transient assay. If it works, then you see some nice glowy confocal images. Be careful, though, as the tagging can affect protein localization.
* Fractionation. In some cases, you can get a rough idea by e.g. extracting nuclei and doing a simple Western blot to see where the protein shows up.
In the real world, you might start with the FP-tag and see that your protein is absent from the nucleus in your mutant. Which would be cool, and interesting in terms of figuring out it's function. If that was presented as the only result, I'd reject the paper and think the authors are terrible investigators. I'd want to see, at least, some nuclei preps that detect the protein in the WT, and don't in the mutant. I'd love to see immunos, too, as FP often does mess up the localization.
You can take it even further and start doing deletions. You take the protein and crop bits out to see what happens. You should see stuff like removing the NLS makes it stop going to the nucleus. That's a good sanity check and a sign your methods are working. You can also try to mess with active sites, protein-protein interaction domains, etc. etc. All within a theoretical model of what you think is going on in the cell.
Ultimately, the difficulty of replication isn't that troublesome. An inability to do so is, but science has never been easy. That's what you sign up for, and that's why you need to read papers critically. You get a sense of distrust for data unless there's really solid evidence of something. And when you find that solid evidence, you get a big smile and warm feeling in nerd heart.
Let's say you want to know where a protein localizes in a cell, of a given tissue, in both mutant and wild-type organisms.
* Immunolocalization. You develop antibodies to the protein of interest, fix and mount tissue, perfuse it with the antibody, and use a secondary antibody to make it detectable.
* Fluorescent tagging. You make a construct with your protein fused to a fluorescent protein. Usually this involves trying a few different tagging strategies until you find one that expresses well. Then you can make a stable transgenic, or try a transient assay. If it works, then you see some nice glowy confocal images. Be careful, though, as the tagging can affect protein localization.
* Fractionation. In some cases, you can get a rough idea by e.g. extracting nuclei and doing a simple Western blot to see where the protein shows up.
In the real world, you might start with the FP-tag and see that your protein is absent from the nucleus in your mutant. Which would be cool, and interesting in terms of figuring out it's function. If that was presented as the only result, I'd reject the paper and think the authors are terrible investigators. I'd want to see, at least, some nuclei preps that detect the protein in the WT, and don't in the mutant. I'd love to see immunos, too, as FP often does mess up the localization.
You can take it even further and start doing deletions. You take the protein and crop bits out to see what happens. You should see stuff like removing the NLS makes it stop going to the nucleus. That's a good sanity check and a sign your methods are working. You can also try to mess with active sites, protein-protein interaction domains, etc. etc. All within a theoretical model of what you think is going on in the cell.
Ultimately, the difficulty of replication isn't that troublesome. An inability to do so is, but science has never been easy. That's what you sign up for, and that's why you need to read papers critically. You get a sense of distrust for data unless there's really solid evidence of something. And when you find that solid evidence, you get a big smile and warm feeling in nerd heart.