Optimize blocking time and/or temperature. Increase the concentration of protein in the blocking buffer. Insufficient blocking of nonspecific sites Use our blocking buffer selection guide to find the most compatible blocking buffer for your experiment. When using an alkaline phosphatase (AP) conjugate, a blocking buffer in Tris-buffered saline (TBS) should be selected because phosphate-buffered saline (PBS) interferes with AP activity. Test for cross-reactivity in blocking buffer by blocking a clean piece of membrane, incubating with antibodies, and then detecting with the substrate of choice. Instead, block with BSA in Tris-buffered saline. When probing for phosphoproteins, avoid phosphate- based buffers like PBS and phosphoprotein-containing blockers like milk or casein.
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Milk contains biotin, which will result in high background. High concentration of RIPA (radioimmunoprecipitation assay) buffer results in widening of lanes and significant streaking during electrophoresisĭilute samples before electrophoresis to lower the final concentration of lysis buffer to prevent buffer-related defects.ĭecrease concentration of primary and/or secondary antibody.ĭo not use milk with avidin–biotin system. Use detergent removal columns or the Thermo Scientific Pierce SDS-PAGE Sample Prep Kit to remove excess detergent. Keep the ratio of SDS to nonionic detergent at 10:1 or greater to minimize these effects. Most of the nonionic detergents (e.g., Triton X-100, NP-40, and Tween 20 detergents) interfere with SDS- polyacrylamide gel electrophoresis (SDS-PAGE). High detergent concentration (e.g., SDS or Triton X-100 detergent) in gel electrophoresis.ĭetergents form mixed micelles with the anionic detergent SDS in the gel and migrate down into the gel they interfere with the SDS–protein binding equilibrium Make sure that the salt concentration does not exceed 100 mM. Use small-volume concentrators such as Pierce Protein Concentrators PES, 0.5 mL. Use a small dialysis device such as the Slide-A-Lyzer MINI Dialysis Device, 0.5 mL.Ĭoncentrate and resuspend samples in lower-salt buffer prior to electrophoresis. Perform dialysis to decrease salt concentration. High salt concentrations result in increased conductivity, which affects protein migration and can result in protein bands spreading into adjacent lanes containing samples with normal salt concentrations Additional data should be considered for a more accurate adjustment.Īpproximately 0.1~0.4 mg/ml of each protein in the buffer (20 mM Tris-phosphate, pH 7.5 at 25☌), 2 % SDS, 0.2 mM Dithiothreitol, 3.6 M Urea, and 15 % (v/v) Glycerol.Excess salt (sodium chloride) in sample during gel electrophoresis. Note: The molecular weight of each protein (kDa) was measured against an unstained protein ladder in every electrophoresis condition.
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2.5 μl per well for general Western transferring.
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Refer to the Blu12 (BLUeye) Prestained Protein Ladder patterns in various electrophoresis conditions:ģ μl or 5 μl per loading for clear visualization during electrophoresis on 15-well or 10-well mini-gel, respectively. Guide for Molecular Weight Estimation (kDa) The Blu12 Prestained Protein Ladder (BLUeye) keeps track of the size and separation of proteins during SDS-polyacrylamide gel electrophoresis, approximating protein size and validating Western transfer efficiency on PVDF, nylon, or nitrocellulose membranes. The 12 recombinant proteins are covalently coupled with blue chromophore, while 1 green band at 25 kDa and a red band at 75 kDa serve as reference bands. The Blu12 Prestained Protein Ladder (BLUeye) is a combination of 12 pre-stained proteins with molecular weights from 11 to 245 kDa.