Protein is Overdrive and OVD & Genes are overdrive and ovd

Far Western Blot Method:

  • OVDs were quantified and separated by SDS or native PAGE.
  • After the separation, OVDs were transmitted from gel to a membrane.
  • The transferred OVDs were denatured and renatured so their binding motif can be completely exposed.
  • The membrane was blocked with BSA buffer.
  • Then, the membrane was incubated with purified OVD baits.
  • The detected signals of the OVD baits were observed and recorded.
  • Lastly, the analyzed images (result)  were recorded as data along with how they were delivered (Far-Western Blot Analysis, 2008-2020).

The method of gel electrophoresis is used before hand to separate the OVD from the ovd mutant followed by the transfer of the membrane in a blotting process. Western Blot is then used with specific antibody probe to identify the OVDs in the sample. After the identification of the OVDs, Far Western Blot implements non-antibody protein that will probe OVDs which is of interest on the blot. It has been stated that the probe OVDs is produced in E. coli while using an expression cloning vector. The denaturing and renaturing of OVDs are exercised. Then the membrane is blocked and then probe with filteredOVD. Furthermore, the membrane exposes spots with OVDwhere the ovd is stationed if both OVD and ovd mutants form a complex molecule. In sum, the OVD cells can be visualized via the method of Immunofluorescence. This method is effective in identifying the interaction that does not necessary entails the native folded protein structure (Far Western Blot Protocol, 2007-2020). [cn1] 

Immunofluorescence[cn2]  Method:

Materials required

  • PBS
  • Paraformaldehyde
  • Methanol
  • Distilled water (dh2O)
  • PBST: 1X PBS, 0.1% Triton X-100. To prepare 1L, add 100ml 10X PBS to 900ml dH2O. Add 1ml Triton X-100 and mix.
  • Primary antibody
  • Fluorochrome-conjugated secondary antibody
  • Mounting Medium

Specimen Preparation

  • The coverslips were treated with a 1:10 dilution of poly-lysine solution at room temperature for 5 minutes.
  • OVD expressive ovarian cell were plated at appropriate dilution and grow until cells reach desired confluence (~70%)
  • The media was articulated.
  • The OVD expressive ovarian cells were rinsed briefly in PBS.

Fixation Step

  • PBS was articulated and OVD expressive ovarian cells were covered with 2-4% formaldehyde in PBS (work in fume hood). Cells could be fixed for 15 minutes at RT.
  • PBS was rinsed for 3X for 5 minutes each.

Blocking Step

  • The OVD expressive ovarian cells were blocked in 5% normal serum from same species as secondary antibody in PBS,1%Triton X-100 for 1 hour.

Primary Antibody Incubation

  • The main antibody was diluted in PBS 0.1% Triton. Typical volumes are: 50-100ul per section, 25-50ul per coverslip, chamber, or well (48 or 96 well plate).
  • Then, the blocking solution was extracted.
  • After blocking, the primary antibody was diluted.
  • The diluted antibody was incubated overnight at 4°C with gentle agitation or rocking.
  • PBS was repeatedly rinsed (3x), 0.1% Triton X-100 for 5 minutes each.

Secondary Antibody Incubation

  • Secondary antibody was incubated with fluorochrome-conjugated secondary antibody diluted in PBS, 0.1% Triton X-100 for 1-2 hours at RT in dark. (From this point on, slides need to be kept in the dark.)
  • PBS was rinsed in 0.1% Triton X-100.

Fluorescence Detection

  • Coverslips were mounted.
  • The resulted sample was examined using fluorescence microscopy immediately or store flat at 4°C in the dark.

Far Western Blot Protocol. (2007-2020). Retrieved from Sino Biological: Biological Solution Specialist: https://www.sinobiological.com/category/far-wb-protocol

Far-Western Blot Analysis. (2008-2020). Retrieved from Creative Proteomics: https://www.creative-proteomics.com/services/far-westernblot-analysis.htm

Immunofluorescence General Protocol. (2015). Retrieved from USBiological: Life Sciences : https://www.usbio.net/protocols/immunofluorescence


 [cn1]Far western is a technique to determine protein-protein interactions. Here you seem to be proposing that OVD protein binds to the ovd gene, which wouldn’t be detected by Far western. ChIP could be used to test this hypothesis. A good hypothesis for Far wester would be that mutated OVD binds to a protein and alters its role in cancer. Your expected results would be to see an interaction between OVD and this other protein.

-6

 [cn2]This looks like a good protocol for IF on cultured cells. IF can be used to detect where proteins are expressed and can be used to quantify relative expression levels. A good hypothesis for this technique could be that OVD expression is greatly increased in cancer compared to healthy cells. Your expected results would be that OVD fluorescence would be brighter in cancer cells compared to healthy cells.

-6