b'Broad range Broad rangeGood to knowkDa kDa kDa kDa How can protein transfer efficiency and ~140 ~198 ~250 consistency be assessed?-~115 ~180 ~98 ~148~80 ~130Visible light Near-IR and RGB detectionVisible light Near-IR and RGB detection ~70 ~100 ~62 ~98 Transfer efficiency refers to the efficiency of ~50 ~70 ~49 ~64 transfer of protein out of the protein gel and onto ~40 ~55 ~38 ~50 the western blotting membrane. Specifically, the ~30 ~40 ~28 ~36 amount of protein that binds to the membrane will ~25 ~35~25 ~17 ~22 be the foundation for the immunoblotting steps. ~15 ~14 ~16 If very little protein transfers out of the gel and ~15 ~6 ~6 binds to the membrane, then the subsequent ~10 ~10 ~3 ~4 immunoblotting steps will be challenging.NuPAGE Novex NuPAGE Novex Transfer efficiency is sometimes evaluated by 412% Bis-Tris 420% Tris-Glycine Gel 412% Bis-Tris Gel420% Tris-Glycine Gel Gel (MES bu\x07er) (MES buer) visually assessing the amount of prestained protein ladder that is visible on the membrane Thermo Scientific PageRulerSeeBlue Plus2 post-transfer. However, this is not necessarily Prestained Protein Ladder Prestained Protein Standard the best practice. Prestained ladder proteins are Cat. No. 26616 Cat. No. LC5925 stained with dyes that allow for visualization during electrophoresis. These dyes affect the transfer High rangeHigh range of the ladder proteins out of the gel. Sample proteins, however, are not stained and thus can kDa kDa have a different mobility rate out of the gel.Visual assessment of the transfer of ladder can be ~460 a useful quick process check for whether transfer ~270 ~268Visible light Near-IR and RGB detectionVisible light Near-IR detection~185 ~238 occurred (sometimes electrodes may be reversed, ~140 ~171 or the transfer stack may not be assembled ~115 ~117 correctly). Not observing any prestained marker ~80 ~71 transfer to the membrane could indicate an ~65 issue with the transfer setup. A more favorable ~50 ~55 strategy to determine transfer efficiency is to use ~40 ~41~31 a reversible membrane stain, which will stain the total protein that successfully transferred to NuPAGE NuPAGE the membrane. Some choose to take this a step 38% Tris-Acetate Gel38% Tris-Acetate Gel further and back-stain the gel, post-transfer, which can give an assessment of how much protein Thermo Scientific Spectra Invitrogen HiMark transferred out of the gel. It is not unusual to see Multicolor High Range Protein Ladder Prestained Protein Standard ladder protein or sample protein remaining in the Cat. No. 26625 Cat. No. LC5699 gel, as no transfer method is completely 100% efficient. What really matters is how much protein is bound to the membrane.Transfer consistency refers to the evenness of protein transfer from gel to membrane. Ideally, protein is transferred evenly from side to side Good to know and top to bottom on the membrane. However, improper assembly of a transfer stack and failure Why do molecular weights change between the differentto roll the transfer stack may lead to air bubbles, gel chemistries? creases, and dead spots on the membrane. These Slight differences in protein mobilities will occur when the sameartifacts make the subsequent immunoblotting proteins are run in different SDS-PAGE buffer systems (e.g., Bis-Trissteps challenging, as an uneven transfer of vs. Tris-glycine). Each SDS-PAGE buffer system has a different pH,protein will lead to an uneven immunoblot and which affects the charge of a protein and its binding capacity for SDS.signal production. Transfer consistency cannot be The degree of change in protein mobility is usually small in naturalassessed simply by the appearance of transferred proteins but is more pronounced with atypical or chemically modifiedprestained ladder proteins on the membrane. proteins, such as prestained standards. Apparent molecular weightInstead, the use of a reversible membrane stain values for prestained standards will vary between gel systemsit isprovides a more accurate picture of protein important to use the apparent molecular weights that match your geltransfer efficiency across the entire membrane.for the most accurate calibration of your sample proteins.48'