They bind to specific substrate s and provide the optimal conditions in their "active site" for a reaction to occur. So, after they help out with some reaction, they can do it again and again and again. I say "help out" because the enzyme doesn't really "do" anything - it can only help make something that was "possible," "likely. Instead, enzymes lower the activation energy required to get the reaction going.
Plus, it increases sensitivity because multiple secondary antibodies can bind each bound primary one so it amplifies the signal. The heat gives the molecules enough energy to overcome the weak interactions that give the protein chain its overall fold, but not enough energy to break the chain itself.
As it unfolds, the SDS slithers in and coats the protein. And the SDS is negatively charged, so it makes the protein uniformly negatively charged and prevents refolding because thanks to that like charges repel thing it basically makes the whole protein hate itself. But the negative charge makes it love the positive charge you stick at the bottom of the gel!
You can use the negative charge to direct the protein to travel through a polyacrylamide gel mesh towards a positively-charged electrode. Longer proteins get tangled up more so travel more slowly. An appropriate buffer is added to create a charge gradient upon the application of an electric current.
And since the gel can heat up when a charge is applied, the buffer helps to keep the gel cool and prevent it from overheating. Since proteins and nucleic acids both have a uniformly net-negative charge, these molecules will naturally migrate towards the positive electrode upon the application of an electric current. Since smaller molecules can easily move through the pores of the gel matrix, you can expect them to migrate through the gel matrix faster than the larger molecules.
Basically, there are two main types of gel electrophoresis that you can use for your application - horizontal gel electrophoresis and vertical gel electrophoresis. While both systems follow a similar theory of gel electrophoresis, there are some key differences between them. How do these systems differ and when should you choose one over the other?
Orientation and Buffer System. Note that phospho-specific antibodies may react with a milk blocking agent due to the presence of the phosphoprotein casein. If using phospho-specific antibodies, block with BSA instead of milk. Residual unbound antibodies or other reagents remaining between steps can produce a high background. If using fluorescent detection, be sure to remove Ponceau S before immunostaining as this can autofluoresce.
Reduce the amount of signal amplification eg conjugate less biotin to secondary antibody if using biotinylation. Nitrocellulose membranes generally give less background than PVDF; consider using a nitrocellulose membrane instead if high background persists. This is likely if you see extra bands at high molecular weights that are 2x or 3x the weight of the expected bands. Some proteins will form dimers, trimers, or larger multimers due to disulfide bond formation if the samples are insufficiently reduced.
To prevent this, try boiling the sample for longer in Laemmli buffer during sample preparation. Many proteins display bands at slightly higher molecular weights than expected due to post-translational modifications PTMs such as phosphorylation and glycosylation or alternative splice variants.
Check the literature to see if multiple bands are reported. To confirm the extra bands are due to PTMs, you may break down modified proteins by treating samples with suitable reagents. For example, PNGase F can remove glycosylations. The additional bands should then disappear when running another blot. Cell lines that have been frequently passaged gradually accumulate differences in their protein expression profiles Go back to the original non-passaged cell line and run these samples in parallel.
Where possible, use blocking peptides to differentiate between specific and non-specific bands. Only specific bands should be blocked and thus disappear. Make sure you use fresh, sterile buffer eg our sterile PBS. Bubbles will appear as uneven white spots. Make sure you remove any air bubbles caught between the gel and the membrane during transfer.
You can do this by lightly pressing down on the stack with a small roller. Before proceeding with blocking and immunostaining, check the transfer of proteins to the membrane with Ponceau S.
If all bands appear very low, you may have left the proteins too long to migrate through the gel. This is because the proteins do not experience enough resistance, so migrate too quickly across the gel.
You should generally run lower molecular weight proteins in gels with a higher percentage of acrylamide. Check this table for suggested gel recipes, and increase the amount of acrylamide if necessary. If all bands appear very high, the proteins may not have had enough time to migrate across the gel. This is because a high acrylamide density can block effective migration of proteins through the gel.
Check this table for suggested gel recipes, and reduce the amount of acrylamide if necessary. If the voltage is too high, migration will occur too quickly. Check the protocol for the suggested voltage and decrease if necessary. If the temperature is too high, the pH of the buffer may be slightly altered, which could affect migration. When the gel has not polymerized properly, bands can appear wonky or uneven. In extreme cases, lanes probed for the same protein can appear at different molecular weights see image above.
See our suggested gel recipes here. If the antibody concentration is very high, then the substrate is consumed very quickly. This means very little light is absorbed at this point, leading to a white band when you image the blot. Use enzymes to remove suspected modification returning molecular weight closer to expected Check amino acid sequence and literature. Dimers, multimers, or protein-protein interactions may be occuring because samples have not been fully reduced and denatured. Primary antibody concentration may be too high, or there is a cross-reactivity with similar epitopes on other proteins.
Use an affinity-purified primary antibody Optimize primary antibody concentration Try another antibody Check antibody specificity with blocking peptide. Carefully remove air bubbles between the gel and the membrane before protein transfer. White negative bands on the film when using ECL detection.
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