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Product Tips | Application Tips |
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| If you are using the ReadyPrep 2-D starter kit and have extra sample rehydration buffer, you can store the buffer at –20°C or lower (preferably –70°C to prevent urea breakdown) for 3–4 months. Store the buffer in aliquots and avoid freeze-thaw cycles. |
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| If you are using the Rotofor, mini Rotofor, or MicroRotofor cell, and all of the proteins in your sample are focusing at one end of the sample chamber, consider using nonionic or zwitterionic detergents. Protein samples that contain an anionic detergent, like SDS, or strong reducing agents, like beta-mercaptoethanol, may acquire a net charge that causes them to experience a shift in apparent pI and migrate to one end of the cell during isoelectric focusing. |
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| You can keep the equilibration buffer I from the ReadyPrep 2-D starter kit for 3–4 months if you store it at –20°C. |
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| The ReadyPrep 2-D starter kit is optimized for use with the pH 4–7 ReadyStrip IPG strips. If using the pH 3–10 strips, apply a quarter of the recommended amount of sample protein in the same recommended volume of rehydration buffer during the rehydration step. |
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| If you are using the ReadyPrep sequential extraction kit, you can store an opened ampule of tributylphosphine (TBP) for up to 6 months. Store the TBP at –20°C in a tightly sealed glass vial with a Teflon-coated lid. |
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| When using the ReadyPrep sequential extraction kit, do not substitute tris (2-carboxyethyl) phosphine (TCEP) for tributylphosphine (TBP). TCEP carries a strong negative charge and migrates to the anode during isoelectric focusing. This leaves proteins in the neutral and basic areas of the IPG strip unprotected from reoxidation and increases the time required for focusing. |
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| For best results with the ReadyPrep sequential extraction kit, wash the pellet resulting from each step. Each time, resuspend the pellet in the solution previously used for extraction and repellet the sample. |
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| When using the Model 491 prep cell or mini prep cell, bear in mind that protein peaks are typically not visible on the elution profile of a UV monitor. To locate the fractions containing the protein of interest, analyze every third tube on a mini gel and apply a sensitive stain, such as the Silver Stain Plus kit. |
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| Concentrate fractions from the Model 491 prep cell or the mini prep cell by dialysis against purified, high-mass polyethylene glycol, spin filtration, or lypholization. Proteins can also be concentrated by retention on a small column followed by elution with a small volume of buffer. |
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| The ideal flow rates for the elution buffer on the Model 491 prep cell and mini prep cell are 0.5–1 ml/min and 75–100 µl/min, respectively. However, optimize your gel with a flow rate of 1.0 ml/min for the Model 491 prep cell and 100 µl/min for the mini prep cell before testing different flow rates. In general, faster flow rates generate more dilute protein fractions and yield better resolution as they distribute the different sample components over more tubes. |
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| When performing a particularly difficult separation on the Model 491 prep cell or mini prep cell, run the sample on a short prep cell gel first, then concentrate the fractions containing the protein of interest and run the sample again on a fresh prep cell gel. This procedure should eliminate most of the proteins that may interfere with your separation. |
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| You can reuse the buffer used in the lower chamber of the Mini-PROTEAN 3 Dodeca cell up to 10 times. After the tenth use, the pH of the buffer may change and any species present in the chamber may migrate into the gel during electrophoresis, causing problems with staining. |
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| For best results, do not reuse a Model 491 prep cell or mini prep cell gel. Large proteins in the sample remain on and move slowly through the gel. During subsequent runs, these proteins elute as a continuous background of very broad bands, such that every fraction contains this background of contaminating protein. |
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| Store the E. coli sample from the ReadyPrep 2-D starter kit in aliquots at –20°C or lower for 3–4 months. After that time, some proteins will be lost to precipitation. If you use the sample after 3–4 months, spin it down before applying it to the IPG strip. Following long-term storage, the spots will not be as sharp as when the sample was first prepared. |
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| When using protease or phosphatase inhibitors with any of the ReadyPrep sample preparation kits, add the inhibitors during the lysis step of extraction. |
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| Leakage of acids or bases into the sample focusing chamber of the Rotofor system both decreases the number of fractions on the linear portion of the pH gradient and reduces the effective voltage across the sample. To determine if this is occurring, fill the Rotofor focusing chamber with deionized water and run it at 12 W constant power. The amperage should decrease to <6 mA and the voltage should increase to 2,000 V within several minutes. If it does not, electrolyte is likely leaking into your sample. |
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| Store ReadyStrip IPG strips after use by removing them from the focusing tray and placing them in a rehydration tray with the gel side up. Do not attempt to remove any oil remaining on the strips. Wrap the strip tightly in plastic wrap and store at –20°C. |
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| Prior to using ReadyStrip IPG strips, remove small ionic molecules from your sample using the Micro Bio-Spin 6 or Micro Bio-Spin 30 columns. These columns contain 10 mM Tris and remove all the small molecules from protein samples with excellent protein recovery and no dilution. |
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| The PROTEAN IEF cup loading tray often improves the resolution of proteins with pIs in the acidic or basic range when compared to loading proteins by in-gel rehydration. Place the loading cup at discrete positions along the strip to optimize individual protein focusing. |
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| When using the cup loading tray with the PROTEAN IEF cell, rehydrate the IPG strips for 12–16 hours before placing the sample cup on them for sample application. |
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| Use the ReadyPrep reduction-alkylation kit to focus basic proteins. This kit reduces and alkylates proteins, effectively blocking thiol groups to prevent re-oxidation during focusing. |
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| Proper cleaning of the PROTEAN IEF cell trays eliminates wicking (mineral oil migration between lanes). Use hot water with detergent and rinse the tray thoroughly. Any residual soap left in the tray worsens wicking. When washing and rinsing the trays, pay special attention to the corners. |
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| The PROTEAN IEF cell does not recover and continue to run after a power failure. If your laboratory is susceptible to power failures, use an uninterruptible power supply for this and other electronic instruments. |
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| Clean the cup loading tray and movable electrodes of the PROTEAN IEF cell after each use with a soft-bristled brush, hot water, and detergent to remove residual mineral oil and sample solution. Do not use organic solvents on either the focusing tray or movable electrodes. Discard sample cups after use. |
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| When using the cup loading tray on the PROTEAN IEF cell, use at least two sample cups to ensure a precise fit of the cup to the strip. |
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| After gel casting, wash the Mini-PROTEAN Tetra spacer plates with a laboratory detergent, rinse with deionized water, and dry them. Do this immediately after use, and do not leave the plates to soak overnight. |
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| When using the Mini-PROTEAN Tetra spacer plates, limit submersion in strongly basic solutions, such as >100 mM NaOH, to less than 24 hours. Also limit submersion in chromic-sulfuric acid glass cleaning solution to 2–3 hours. Prolonged submersion compromises the integrity of the adhesive that holds the spacers on the plate. |
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| Though not recommended, it is possible to reuse Flamingo fluorescent gel stain. The sensitivity of the stain and the image quality will, however, decrease after the first use. |
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| Store gels that are stained with Flamingo fluorescent gel stain at 4°C and protected from light for up to 6 months. Store the gels with 5–10 ml of staining solution in a sealed container or sealable plastic bag. |
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| Use glass containers when staining gels with Flamingo fluorescent gel stain, as they can be cleaned more thoroughly than plastic containers. High or uneven background staining may result from using plastic trays that have not been cleaned and rinsed completely. The use of plastic containers does not affect the sensitivity of detection, though. |
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| To achieve proper staining with Bio-Safe Coomassie stain, wash the gel as directed on the label of the stain. SDS and other electrophoretic contaminants that may be present in the gel can interfere with the staining process, which results in little to no stain. To prevent this, wash the gels thoroughly with three water washes before staining and take care to remove all the water from the last wash before adding the stain. |
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| If using Bio-Safe Coomassie stain to analyze peptides, fix the peptides with 40% methanol and 10% acetic acid for at least 30 minutes before applying the stain. For a more stringent fixation, use 40% methanol and 10% trichloroacetic acid for 30 minutes; this method should be the most effective at fixing peptides to a gel. In any case, fix, stain, and destain the peptide gel for the minimum effective time at each step to minimize loss of peptides from the gel. |
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| When using the Silver Stain Plus kit, add the development accelerator quickly, while it is stirring vigorously at room temperature, to avoid the formation of a white, cloudy precipitate. |
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| Store gels stained with SYPRO Ruby in the stain, protected from light, for up to one month without any substantial decrease in signal. |
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| Flamingo fluorescent gel stain is compatible with mass spectrometry applications and yields better sequence coverage and protein identification than SYPRO Ruby protein gel stain. |
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| When staining an acrylamide gel with Coomassie Blue, SYPRO Ruby, Flamingo, or silver stain, you can substitute ethanol for methanol in the fixing solution. Use a slightly lower concentration of ethanol (30–35% SDA 3A ethanol denatured with methanol and isopropanol) than is required for methanol (40%). |
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| Clean the PROTEAN II xi cell with any common laboratory detergent. The cell is not compatible with organic solvents such as chlorinated hydrocarbons (for example, chloroform), aromatic hydrocarbons (for example, toluene or benzene), acetone, ethanol, or 2-amino-2-methyl-1, 3 propanediol. |
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| When using the PROTEAN II xi cell, make sure the sample is denser than the surrounding buffer to prevent the sample from floating out of the well. Make sure the sample contains about 10% glycerol. Do not load the sample into the well too quickly, as it may bounce off the bottom or sides and land in another well. |
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| You can store a prep cell acrylamide gel in the refrigerator before use for up to 4 days. When doing so, cover the gel with an overlay of gel buffer (at the same concentration as in the gel) and with sealing film. |
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Application Tips | Product Tips |
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| When working with membrane or insoluble proteins, increase the amount of SDS in the equilibration and running buffers (up to 0.2%) to allow the proteins to effectively migrate out of the IPG strip. |
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| Generally, the best method for keeping a protein in solution is to add any combination of nonionic detergents, zwitterionic detergents, and chaotropic agents to the sample mixture. Also use reducing agents such as DTT and DTE (less than 20 mM) to decrease disulfide bond formation between proteins. |
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| To reduce the amount of SDS in samples generated by preparative SDS-PAGE, substitute the elution buffer with one that does not contain SDS. |
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| Nucleic acid contamination is a common cause of horizontal gel streaking. Treat samples with nucleases to remove nucleic acids prior to isoelectric focusing. |
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| Never heat samples in urea-containing buffers. The urea rapidly breaks down to carbamic acid and carbamylates the proteins, modifying their charge. Urea breakdown and subsequent protein carbamylation is the cause of charge trains on 2-D gels. A charge train is a series of spots on a 2-D gel that are of different pIs and the same size. |
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| Negatively charged polysaccharides that contain sialic acid can produce horizontal streaks similar to those generated by nucleic acid contaminants. Ultracentrifugation is often sufficient to remove carbohydrates from samples. |
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| To prevent vertical streaking, limit the amount of protein added onto an IPG strip. Compensate for such decreases in sample load by using a more sensitive staining technique, such as silver staining. |
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| Reusing electrophoresis running buffer can result in poor separation and vertical streaking due to the depletion of ions and SDS in the running buffer. Avoid this practice, especially if vertical streaking is a persistent problem. |
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| Vertical streaking on second-dimension gels is often caused by gaps between the IPG strips and the gels. Ensure the second-dimension gel has a straight and level top edge, and that the IPG strip is in direct contact with the gel along its entire length. |
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| If some of the bands on your gel are not staining or appear faint, use silver stain as usual, then agitate it slowly in deionized water for 30 minutes and repeat. Then apply the silver stain again, starting with the silver reagent step. Proteins that did not stain on the first cycle will stain to full intensity. |
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| Vertical streaking is often caused by overfocusing — isoelectric precipitation (pI fallout) increases with focusing time. For this reason, do not conduct first-dimension IEF for any longer than is necessary. |
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| To ensure proper and consistent visualization with a silver stain, use ultrapure water with all organic contaminants removed for the final rinse of your staining vessel. In addition, reserve that vessel exclusively for silver staining, and separate it from other glassware in your laboratory. |
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| Proteins must be soluble if they are to be separated and identified on 2-D gels. Protein insolubility (precipitation) leads to loss of sample spots and streaking on 2-D gels. |
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| Fractionation may improve your 2-D result by reducing sample complexity, improving the range of detection, and enriching low-abundance proteins. Fractionation can be performed according to many protein properties, including subcellular location, solubility, size, charge, and pI. |
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| Improve the resolution and reproducibility of 2-D gels by performing sample cleanup to remove salts, charged detergents, phenolics, lipids, sugars, and nucleic acids. Cleanup will also reduce disulfide bonds. |
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| NaCl increases conductivity, extends the time required for focusing, causes electroendosmosis, and results in uneven water distribution in the gel. In general, the tolerated concentrations of NaCl for proper in-gel rehydration and cup loading are 10 mM and 40 mM, respectively. |
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| Nucleic acids bind proteins through electrostatic interaction, thereby interfering with isoelectric focusing. Nucleic acids can also clog the pores of the acrylamide matrix. Remove nucleic acids with nucleases and ultracentrifugation in the presence of carrier ampholytes. In addition, benzonase can be used in a sample together with urea to remove DNA or RNA contamination. |
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| Insoluble material in a sample obstructs gel pores, resulting in poor focusing and severe streaking. Remove these materials by high-speed centrifugation (for example, 20,000 x g for 30 minutes at 20°C). |
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| To monitor the initial progress of the electrophoresis on the IPG strips, add up to 0.001% of Bromophenol Blue to both the rehydration and equilibration buffers.
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