Quantitative Western Blot (1D)

Our quantitative 1D Western blot service, with Fluorescent labeled protein samples, offers the following unique features:

  1. High sensitivity: 1ng /band
  2. Accurate quantitation: Band intensity and band percentage by ImageQuantTM TL (GE Healthcare, version 7.0)
  3. Multiplex Western blot: 1 or 2-color Western blot can detect 1 or 2 antibody in a single blot

Example 1: Two-color Western blot detecting time-dependent phosphorylation of 14-3-3

Total proteins were extracted from the drug treated HT29 cells at different time points. In each lane, equal amount of protein was labeled and loaded. The gel was then transferred to a PVDF membrane followed by a 2-color Western blot. The data shows that while the total protein level of 14-3-3 (B. Anti-14-3-3 antibody) did not change, the phosphorylated 14-3-3 (C. Anti-phospho14-3-3 antibody) clearly increased during the time course.

  • A: Total proteins
  • B: Western blot with Mouse Anti-14-3-3 antibody
  • C: Western blot with Rabbit Anti-phospho14-3-3 antibody

A. HT29 Cell Total Protein

Quantitative 2-color Western Blot: drug treated HT29 cell lysate at different time points

B. Mouse Anti-14-3-3

Quantitative 2-color Western Blot: HT29 cell lysate blotted with Anti-14-3-3 antibody

C. Rabbit Anti-phospho 14-3-3

Quantitative 2-color Western Blot: HT29 cell lysate blotted with Anti-phospho 14-3-3 antibody

B+C. 2-Color-WB Overlay

Quantitative 2-color Western Blot: overlay image of HT29 cell lysate blotted with Anti-14-3-3 (green) and Anti-phospho 14-3-3 (red) antibody

A+B. Image overlay

Quantitative 2-color Western Blot: overlay image of HT29 cell lysate (blue) blotted with Anti-14-3-3 antibody (green)

A+C. Image overlay

Quantitative 2-color Western Blot: overlay image of HT29 cell lysate (blue) blotted with Anti-phospho 14-3-3 antibody (red)

A+B+C. Triple-color Overlay

Quantitative 2-color Western Blot: triple overlay image of HT29 cell lysate (blue) blotted with Anti-14-3-3 (green) Anti-phospho 14-3-3 antibody (red)

Sample Info

Please follow these general principles regarding biohazardous materials and buffer condition in getting your samples ready.

Protein amount for each WB: 20-100 µg of total protein from each sample.

Protein concentration: 5-20 mg/ml is preferred.

Primary antibody requirement for each WB: Sufficient for 50ml incubation volume, and need information on:

  • Animal species in which the antibody was raised
  • Suggested dilution factor

If you cannot provide the dilution factor, please let us know:

  • If it is a commercial antibody, provide the link to the antibody information
  • If it is not a commercial antibody, please provide the protein concentration of the antibody, whether it is IgG fraction or anti-sera or antigen-affinity purified.

Buffer: Please feel free to submit samples in whichever buffer that you would normally use. There is a sample prep charge for each sample of 1D Western blot. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.

Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.

Pricing

Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
CodePriceCodePrice
1D Gel (up to 10 samples) 1418A$500418N$600
Transfer to PVDF or Nitrocellulose Membrane 2520A$250520N$295
Western Blot with One Color 3512A$410512N$495
Western Blot with Two Color 3513Acall for price513Ncall for price

Customers are responsible for the primary antibodies, please call Tech Support to discuss details

1. Price does NOT cover sample preparation or CyDye labeling

2. Price covers:

  • Trans-blotting 2D Gel to PVDF or Nitrocellulose membrane
  • Membrane images scanned after the transfer

3. Price covers:

  • Western blot using the primary antibodies provided by customers, Applied Biomics will provide CyDye labeled anti-Mouse/Rabbit/Goat/Sheep secondary antibodies.
  • Membrane images scanned after Western Blot

1-3. Each Western blot will cover 2 hour data analysis:

  • Protein and Western blot images

Price does NOT cover:

  • Primary antibody provided by customer
  • Customized data request in addition to the standard request