Western Blots


One-Step Western Blot analysis flow chartOne-StepTM Western Kit is a new product incorporating GenScript's innovative thinking and proprietary technologies. The One-StepTM Western cuts total procedure time of a Western or Dot blot from 4½ hours to one hour with no loss in sensitivity. A classical three-step Western blot requires blocking, primary antibody binding and secondary antibody binding, which can take 4½ hours. One-StepTM Western users may simply transfer their proteins from gel to membrane, incubate the membrane in One-Step Pretreatment Solution for five minutes and then in WB solution with primary antibody for 40 minutes, followed by three five-minute washes. Then the membrane is ready to be developed.

Key Features:
  • Easy to perform: Quick and simple procedure.
  • High sensitivity: The sensitivity of the One-StepTM Western is comparable to or better than that of the classical 4.5-hour procedure, depending on the quality and quantity of antibodies used.
  • Highly reproducible results.
  • Needs less optimization than the classical method.
  • No secondary antibody needed.
  • No special labeling required for primary antibody.

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One-StepTM IP-Western Kit

Overview of One-Step IP-Western Procedure
Figure 1: Trx fusion protein was immunoprecipitated using anti-Trx (GenScript, A00180) mouse monoclonal antibody bound on Protein G Resin (GenScript, L00209). Trx fusion protein was then immunoblotted with the same primary antibody by using HRP-conjugated goat anti-mouse IgG (Fc specific, Sigma, A 0168) (Lane 1) and using One-Step IP-Western Kit L00232 (Lane 2).
Immunoprecipitation (IP) is a common technique used for identifying protein-protein interactions. IP is usually performed using a primary antibody together with a second antibody resin or Protein A or G resin. After IP, a Western blot is used to detect any possible proteins associated with the target protein.

One problem with IP Western blotting, however, is that the image is always contaminated with heavy chains, light chains or Proteins A or G from the resins. This makes the desired proteins harder to identify.

An innovative new product the One-StepTM IP-Western Kit, completely removes this obstacle. With this kit, the user can apply Protein A, G, A/G or any secondary antibody resin with NO contamination whatsoever from heavy chains, light chains or Proteins A or G.

In addition to eliminating blot contamination, the One-StepTM IP-Western Kit:
  • Brings total IP-Western time down to ONE HOUR.
  • Allows for the use of Protein A, G, A/G or any secondary antibody resin during IP.
  • Needs no secondary antibody.
  • Shows high sensitivity and low background.
One-StepTM IP-Western Kit contains all the necessary reagents, buffers, nitrocellulose membrane and HRP substrate for performing an IP-Western blot. After transferring the proteins from gel to membrane, the user incubate the membrane in Pretreat Solution for five minutes, follwed by incubation in WB solution (prepared from primary antibody, IP-WB 1, IP-WB 2 and IP-WB 3) for 40 minutes. Then the membrane is washed three times for five minutes each. The blot can be developed using the HRP substrate included in the kit. The One-StepTM IP-Western blot result is compared with the conventional western result in Figure 1. >> click here to order

One-StepTM Complete IP-Western Kits including L00231 (for rabbit primary antibody), L00232 (for mouse primary antibody) and L00233 (for goat primary antibody) combines high sensitivity with low background. It detects nanograms of the desired antigen while leaving quantities of antibody as high as 2 μg per lane undetected. The A&G blocker included in the kit takes five minutes to use and gives clean IP-Western results even with serious Protein A, G or A/G carryover contamination. For kit L00231 (rabbit primary antibody), Protein G Blocker is simply added to the IP-WB 3 solution. The protein A carryover does not affect the Western results. >> click here to order

Applications:
  • Protein A, G or A/G Resin.
  • Any secondary antibody Resin.