Opti-Protein Ultra Marker

1. Protein shape and migration change in different buffers:
Proteins can take on slightly different shapes depending on the buffer system (like MOPS, MES, or Tris-Glycine). These shape changes affect how fast they move through the gel.

2. Prestained markers behave differently than unstained proteins:
Our protein markers are prestained, meaning each protein is attached to a dye. This dye changes how the protein moves through the gel. So even though the core protein stays the same size, the protein + dye complex migrates differently than a natural (unstained) protein of the same molecular weight.

3. Molecular weights are calibrated for accuracy:
To make the markers useful, we've calibrated them in each buffer system. That means we adjust the apparent molecular weights (in kDa) so they match how unstained proteins would behave at that same migration point. This way, when you compare your unstained sample to our prestained ladder, the molecular weights still match up correctly.

High background is often caused by insufficient blocking, high antibody concentration, or non-specific binding. To reduce it:

Blocking: Block for at least 30 minutes using 5% non-fat dry milk, 3% BSA, or normal serum. These can also be included in antibody buffers. For phospho-specific antibodies, avoid milk (it contains casein, a phosphoprotein) and use BSA instead.

Antibody Optimization: Use the lowest effective antibody concentration. Incubate longer at lower concentrations to improve specificity.

Incubation Conditions: Perform antibody incubations at 4°C to reduce non-specific interactions.

Secondary Antibody Issues: Secondary antibodies may bind non-specifically or react with blocking agents. Run a secondary-only control to check for this.

Washing: Increase the number and duration of washes, and include Tween-20 in your buffers to help remove unbound antibodies.

Membrane Type & Care: Nitrocellulose typically produces less background than PVDF. Always keep the membrane moist during all steps, as drying can increase background.

1. Cell Line Variation: Frequently passaged cell lines may show altered protein expression. Compare results with the original, low-passage line by running both samples side by side.

2. Post-Translational Modifications (PTMs): Proteins may be phosphorylated, glycosylated, acetylated, etc., causing shifts in size. Use enzymatic treatments (e.g., phosphatases, deglycosylases) to confirm.

3. Protein Degradation: Low molecular weight bands may be degradation products. Use protease inhibitors in your lysis buffer to prevent this.

4. Alternative Splicing or Isoforms: The antibody may detect splice variants or related proteins with shared epitopes. Check the literature and perform a BLAST search for related sequences.

5. High Antibody Concentration: Too much primary or secondary antibody can cause non-specific bands. Dilute your antibodies and run a secondary-only control to check.

6. Antibody Purity: Unpurified antibodies may bind non-specifically. Use affinity-purified antibodies for better specificity.

7. Non-specific Binding: Confirm specificity using a blocking peptide—specific bands should disappear when blocked.

8. Protein Multimers: Your target may form dimers or higher-order structures. Try boiling in SDS sample buffer for 10 minutes to disrupt multimers.