Anti-HA Affinity Matrix Protocol & Troubleshooting

Product No. 11815016001

Protocol

Concentrated Eluate

To concentrate the protein eluate, Roche recommends following the immunoprecipitation protocol given in the package insert from step 2 to 5. Use 50 μl of resuspended Anti-HA Affinity Matrix in a microcentrifuge tube. Take the wash and elution buffer as described in the affinity purification protocol. After 3x washing (step 5 of immunoprecipation protocol) incubate the matrix with 50 μl of elution buffer for 15 minutes at +37 °C for elution.
Note: Elutions may be performed at lower temperatures, however, lower yields of purified protein result. Spin down as before and collect the supernatant. Repeat the elution step two additional times. At the end you will have the protein in a total of 150 μl elution buffer. Even two times elution with 50 μl is sufficient to obtain the most proteins.

Pre-Clearing of Lysate

Preclearing of lysate was not found to be necessary to obtain good immunoprecipitation results with the mammalian, bacterial and yeast extracts tested during development of the product. Nevertheless, any cross-linked agarose should be sufficient for preclearing, though this was not tested.

Troubleshooting

Little or no HA-tagged protein is eluted

  • Tagged protein is degraded.

→ Include protease inhibitors and perform purification at +2 to +8 °C.

  • Tagged protein not fully eluted.

→ If working at less than +37 °C, increase temperature, time, and/or number of elutions. Try batch mixing of peptide solution with matrix.

  • Tagged protein expression is absent.

→ Check for expression of protein in crude extract by western blot or biochemical assay.

  • Tagged protein expression is very low.

→ Load larger volume of extract. Run column several times, pool, and concentrate final eluates.

Large quantities of tagged protein remain in the flow-through sample

  • Column is overloaded.

→ Decrease amount of loaded protein extract.

  • Column not regenerated after use.

→ Regenerate column.

Column flow stops

  • Column is overloaded.

→ Decrease amount of loaded protein extract.

  • Starting extract contains insoluble materials.

→ Preclear starting extract by high-speed centrifugation or filtration.

  • Air bubble in needle.

→ Replace needle or place gentle pressure on column by briefly covering top of column with gloved hand.

Tagged protein appears degraded (a smear or multiple lower molecular weight bands on western blotting)

  • Protease activity during procedure.

→ Increase protease inhibitors in protein extract sample. Perform all steps at lower temperatures.

Tagged protein appears with multiple bands on a silver-stained gel

  • Column is overloaded.

→ Decrease amount of loaded protein extract.

  • Wash step is insufficient.

→ Include detergent in Wash buffer.

Nonspecific reactivity on western blot, especially with high total protein loading

  • Too much tagged protein loaded on gel.

→ Titer down total protein loaded in each lane. Anti-HA High Affinity antibody detects picogram quantities of tagged protein in western blots and less protein needs to be loaded compared with many other antibodies.

  • Crossreactivity from an anti-rat secondary antibody

→ Use Anti-HA-Peroxidase, High Affinity or Anti-HA-Biotin, High Affinity for detection. If an anti-rat secondary antibody is chosen, check carefully for crossreactivity from secondary antibody by running controls using secondary antibody alone.

Diminished reactivity on western blot

  • Inappropriate detection system (e.g., use of an anti-mouse secondary antibody for detection)

→ Choose appropriate detection system.

Influence of Ionic Strength (0.4M Salt)

Antibody-matrix binding will not be affected by 0.4M salt as this is a covalent linkage. It has also not been our experience that antibody-epitope binding is hindered in these salt conditions though different tagged proteins might behave differently. If tagged protein is present, the immunoprecipitation should be successful under these conditions.

Materials

     
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