HI,
I am producing my recombination protein in Fetal Dog kidney cell. Although, It is a transmembrane protein and i am only producing protein for the ectodomain, thus my protein is secreted in media. My expected protein size is 45-55 kDa. My protein has a his tag in its C-terminal end. My work flow is:
1) Collect Media
2) Concentrate the media via Amplicon tube
3. Run the Concentrate through Ni-NTA column to elute only his tag protein
4. Conduct western in 4-12% Bis tris SDS-PAGE Gel and use page ruler pre stain ladder
5. Detect with anti-his tag antibody and antibody against my protein
6. Stain Gel with Gel Code Blue Stain (Invitrogen) (separate gel) and PVDF membrane with Ponsu Stain ( After detecting with antibody )
Problem: Staining provided different bands those are not detected by my both antibodies. I cant see the bands detected by my my antibody while i am sating my gels and PVDF membrane.
I am looking forward for your valuable suggestions.
It sounds like you are Ponceau-staining the membrane after doing the Western blot. This will not work, because the membrane has already been blocked.
Antibodies detect one specific protein (hopefully), even if many proteins are present in the lane. The protein stained by the antibody may be too minor in quantity to see as a band on SDS-PAGE or Ponceau S stained (unblocked) membrane after transfer.
Coomassie Blue staining of SDS-PAGE or Ponceau S staining of a membrane is non-specific, meaning all of the proteins get stained.
So you should not expect to see the same protein pattern with antibody detection of a Western blot as with Coomassie staining of the gel or Ponceau S staining of the membrane before blotting.
Probably you purified very little His-protein so you can visualize it only by western blot. Have you tried to express your ectodomain without the N-terminal-signal sequence?
IMAC purification can yield quite a lot of non-specific binding that appears as extra bands on stained gels. There will be plenty of other metal-binding proteins in the culture supernatant. You need to add imidazole at 20-40 mM to the concentrated media before loading on the column.You also need to add NaCl to 0.3-0.5 M to prevent aggregation of your concentrated protein with other proteins - aggregates will co-purify and cause extra bands to appear. Also adjust the pH to 7.4.
After binding the concentrated medium, you need wash the column with pH7.4 wash buffer containing 20-40 mM imidazole and 0.3-0.5M NaCl. You will need at least 10-20 CV of washing until no protein is detectable in the flow through.
Hopefully these steps will reduce the amount of contaminants co-purifying with your protein of interest.
You might have already known that dye staining will detect all the proteins that are at sufficient levels but WB detects specific proteins and at trace levels not detected by dye staining. Therefore the primary focus may need to shift to expression level. Will you be able to articulate any experiment with trace amounts of the protein that experiences yield losses at every purification step?
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