Table 4. Troubleshooting.

Step	Problem	Possible Reason	Solution
Generation of custom polyclonal antisera.	Peptide yielded no phospho-antibodies/very low number of phospho-antibodies.	The peptide designed had an internal cysteine that was used for conjugation.	An internal cysteine should not be used to conjugate the peptide to hapten for injection, since that will mask the phospho-epitope and result in antibodies that will be very difficult, if not impossible to resolve. Thus, in cases where there may be a cysteine right next to the phospho-epitope, there are better chances of obtaining an antibody of interest by making slightly longer peptides (11-14 mers) and obtaining them from the vendor in a reduced state, such that they do not form internal di-sulfide bonds.
Generation of custom polyclonal antisera.	The sera produced low titer against the phospho-antigen.	The region of the peptide used was low complexity	Anecdotally, we have found that different species of animals sometimes give different results in titer generation. Thus, using more than one animal species may be useful. Or, use 5-10 fold more sera with the same volume of the column (step 5 on) for purification
22	Conjugaton of BSA with peptide did not go to completion.	Inefficient or failed conjugation can occur if the quantity of peptide used is incorrect, if the peptide and/or BSA is not fully dissolved or if a wrong solution is used in the conjugation.	Ensure that the peptide is dissolved completely.
		The peptide did not dissolve efficiently.	Add DMSO to the peptide (at 10%) dissolve. Spin out what has not dissolved. Then re-dissolve this in fresh conjugation buffer, with 10% DMSO and repeat until there is no precipitate left. Pool all the dialyzed samples.
		Conjugation Buffer was not used to dissolve the peptide	Use the conjugation buffer supplied with the kit
		Conjugation Buffer has precipitates	Check the date on which the kit was bought and ensure that it has not been at 4C for too long.
		The conjugation reaction was not allowed to continue for 2 hours at room temperature	Incubate the peptide∷BSA mix at room temperature for 2 hours
22, 41, 64	Conjugation of peptide∷BSA complex did not go to completion/was not in right ratio with Sepharose.	The peptide∷BSA complex was in excess to the Sepharose beads.	Ensure that the peptide∷BSA complex is added in a ration of 2mg of the compelx to 1mg of Sephaorse.
		The peptide∷BSA complex and Sephaorse mix dried in the column.	Ensure that there is enough coupling buffer such that the beads do not dry out.
73, 139	The pH of the collected Glycine eluted antibodies is not neutral.	The 1M Tris used for neutralization was pHed to a pH of 8.	Use 1M Tris that has not been pHed. This allows for the hydroxyl ions to neutralize the strong acidic Glycine solution.
82, 93	There are no IgG fractions upon elution from the phospho-peptide column	Peptide design was flawed, refer to generation of custom polyclonal antisera.
		Instead of using the flow through from the non-phospho peptide column, the elution from the non-phospho peptide column was used for binding to the phospho-peptide column.	Ensure that the FLOW THROUGH from the non-phospho peptide column is the one used to pass over the phospho-peptide column.
		The phospho-peptide column was not washed thoroughly and coupled during preparation with the coupling buffer and wash buffer, resulting in some phospho-peptides coming off the column during antibody elution.	Prepare the phospho-peptide column again, with care taken to follow each step of the protocol.
		The secondary used for blotting is old, and does not work.	Use a positive control sample to ensure that the secondary antibody works.
		ECL kit used was left out at room temperature and the reagent is not optimal.	Use a positive control sample to ensure that the ECL reagent is optimally functioning.
93	The phospho-peptide antibody preparation also recognizes the non-phosph-peptide.	Refer to Figure 1 for possible options for the types of antibodies that can be generated from the phospho-peptide immunogen.	Pass the antibody back over the non-phospho-peptide column, collect flow through that is then passed over the phospho-peptide column.