Exalpha Biologicals, Inc.

Accelerating the Pace of Discovery

Product Highlight

Mouse anti-M13 phage coat protein g8p

Antibodies recognising M13 filamentous phage coat proteins are instrumental in the selection and detection of phages expressing specific antibody fragments or peptide sequences at their surface. The monoclonal antibodies manufactured and supplied by Exalpha react with either the pIII (g3p) or pVIII (g8p) proteins of M13 filamentous bacteriophage. All antibodies are available in a purified format. The antibodies are fully validated and are suitable for a wide range of techniques including:

  • ELISA
  • Flow Cytometry
  • Western Blot
  • Immunohistochemistry
  • Immunoprecipitation
For more information, click here for our M13 Bacteriophage information page.

News

Two more of our excellent products have been published by PubMed:

Potential actionable targets in appendiceal cancer detected by immunohistochemistry, fluorescent in situ hybridization, and mutational analysis
Borazanci, E., et al., J. Gastrointest. Oncol., 8, 164-172 (2017)
Using Exalpha SPARC Antibody (Cat. No. X1867P)

Molecular mechanism underlying the pharmacological interactions of the protein kinase C-β inhibitor enzastaurin and erlotinib in non-small cell lung cancer cells
Steen, N.V., et al., Am. J. Cancer Res., 7, 816-830 (2017)
Using Exalpha's FITC labeled anti PY20 Antibody (Cat. No. X1017)

Immunoprecipitation Protocol

Wash Buffer

20 mM Tris, pH 8.0 containing 0.2% Trition X100

Elution Buffer

Wash buffer containing 4% SDS (and also include protease inhibitors if required)

NOTE: The choice of wash and elution buffers should be empirically determined. The buffers listed here are for general use and will not be ideal for all proteins/systems. If phospho proteins are being pulled down the addition of a phosphatase inhibitor may be required to prevent degradation.

Procedure

  1. Centrifuge the cellular debris in a ultracentrifuge at 100,000 x g for 10 min to remove any non- soluble material that will interfere with later assay.


  2. If sufficient sample is available it is prudent to clear by means of preliminary pass or mock pull down using a pre-column or gel.

  3. Incubate the cleared supernatant with specific antibody against the protein of interest (1 hour is generally sufficient but an overnight incubation may be required for some Ab-Ag interactions). Incubate at 4 degrees C for O.N. or 37 C for shorter periods.

  4. Incubate Sample/Ab mixture with adequate amount of protein A (or G) speharose) 1h at 4 degrees C.

  5. Spin down the beads briefly in a microcentrifuge, 3-5 minutes 1000g, and remove and save supernatant to assay for unbound (depleted) fraction.

  6. Wash beads by adding wash buffer vortexing and spinning down as before. At least three cycles (preferably more) of wash are required to remove any unbound material from gel pellet.

    NOTE: it may be advisable to ad 0.5 M NaCl to the wash buffer to aid in removal of any non-specific binding to the gel.

  7. Spin one final time – remove supernatant and retain pellet.

  8. Add elution buffer to the tube and vortex. Let sit on ice with periodic vortexing for 15 minutes.

  9. Spin down, retain the eluted material /buffer (which will contains you’re your eluted protein of interest/sample. It may be required to repeat the elution step and poll or assay elutions separately as required – depending on avidity of antibody for ligand and protein A/G for your Ab.

  10. Analyze the various fractions obtained in the pull-down assay via Total protein staining or via Western blotting (remember that your sample will contain the monoclonal/polyclonal antibody eluted off the gel and perhaps protein A and G as well – if from a supplier other than Exalpha). If western blotting is performed, it is generally accepted that a separate Ab (not the one used to pull down) should be employed).

  11. A negative control should be included during the immunoprecipitation procedure using beads conjugated to a different antibody to ensure that the proteins co-immunoprecipitated are true positives.