Dear researchers,
I am performing WB of FLAG-Ubl3 HEK cell lysate samples using Anti-Flag antibody magnetic beads for immunoprecipitation of FLAG-Ubl3 by FUJIFILM Wako Co. Ltd. For elution, I have to use Enterokinase to cut Ubl3 from FLAG. However, after performing the WB, I did not get any band of Ubl3 or FLAG. Now, I am uncertain about the functionality of the beads. So, I need to use control such as FLAG peptides. But this peptide is about 1 kDa which cannot be seen in the WB. How can I check the functionality of the beads? Thank you.
Given that your FLAG peptide is too small to be detected on Western Blot (WB).
Use a Positive Control with a Larger FLAG-Tagged Protein :
Expression of FLAG-tagged protein: If you don't already have a larger FLAG-tagged protein to test, you can try expressing a well-characterized FLAG-tagged protein (e.g., FLAG-Flag2, FLAG-Actin, or another control protein with a known molecular weight). This will allow you to assess whether the beads can pull down a FLAG-tagged protein and whether the problem is specific to your FLAG-Ubl3.
Check elution: After the immunoprecipitation, use your Enterokinase to cleave the FLAG tag from the target protein and elute it. You can confirm the presence of the protein on the WB by looking for the correct molecular weight of the cleaved product (for example, FLAG-Actin or FLAG-Flag2). If you see a band after elution, this suggests that the beads are working fine.
Peptide Competition Assay :
To test the beads directly, you can try performing a competition assay using excess FLAG peptide (even though it’s small, this method can help you assess the binding efficiency of your beads).
Procedure: Before performing your IP, pre-incubate your beads with an excess amount of FLAG peptide to "saturate" the binding sites on the beads. After this, if you do the IP, the FLAG peptide will block the binding sites, preventing any FLAG-tagged protein from attaching to the beads.
Results: If the beads are working, the presence of the FLAG peptide should reduce or eliminate the amount of FLAG-Ubl3 or FLAG-tagged control protein detected in your Western Blot.
Bead Loading Control :
Before performing immunoprecipitation, check the beads for non-specific binding. You can perform a "bead-only" control by loading the beads with your lysate but not performing any IP or elution step. After that, run a Western Blot to detect any non-specific binding to the beads. Ideally, this should show very little or no background bands, confirming the specificity of the beads.
Verification of Enterokinase Activity :
Enterokinase Activity Test: Since Enterokinase is used to cleave the FLAG tag, ensure that it’s functional. You can perform a test using a known substrate for Enterokinase (e.g., a synthetic peptide with the Enterokinase cleavage site). After cleavage, run a Western Blot to check for the expected band of cleaved peptide or protein. This will confirm the enzyme's activity in your experiment.
Flow Cytometry or Bead Analysis :
If you have access to flow cytometry or bead-based assays (e.g., Dynabeads), you can check if your beads are still capturing FLAG-tagged proteins using a FLAG-specific antibody for direct bead analysis. This can provide a more rapid and quantitative assessment of bead functionality before running a full WB.
By performing these tests, you should be able to determine whether the problem lies with the beads, the protein of interest, the Enterokinase treatment, or some other step in the workflow. If you confirm the beads are functioning properly with the control FLAG-tagged proteins and still don't get a band for FLAG-Ubl3, the issue may lie in the expression or solubility of FLAG-Ubl3, or potential issues with the Enterokinase cleavage efficiency.
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