We applied the rabbit antiserum to the phosphopeptide column first, then eluted the column and took the eluted fractions to the column of non-phosphopeptide (the flow-through was the used antiserum, which was sent to the client). The eluted fraction of the non-phosphopeptide column will be marked with anti-non-phosphopeptide, but please be aware that those fractions do not only bind to non-phosphopeptide but also bind to phosphopeptide. That's why it reacted preferentially with the phosphoform because it came from the phosphopeptide first. Actually, there should be no real anti-non-phosphopeptide antibody in both flow-through and eluted fractions.

The flow-through of the non-phosphopeptide column did only bind to the phosphopeptide, so it's a real anti-phosphopeptide antibody. In our experience, it's better to re-purify this fraction by the phosphopeptide column to get a specific anti-phosphopeptide antibody. The eluted fractions of the re-purification with a phosphopeptide column are the final purified antibodies. These two should be exactly the same.

To get the real anti-non-phosphopeptide antibody, the antiserum must be applied to the non-phosphopeptide column first, and then the non-specific IgG must be removed by the phosphopeptide column. We can do this purification with the used antiserum. The price is $700.

We did provide you with an antibody ELISA test against phospho- and non-phospho-peptides; it will be upon you to test with the corresponding phospho- and non-phospho-proteins by your application, for example, by Western blot. But bear in mind, it's very unpredictable if a designed peptide antibody is against the native protein. There is a gap between peptide and whole protein due to conformation and other structural differences, for example, glycolysis. But we have had experience that some of our clients phospho-antibodies did work nicely in immunohischemistry; the antibodies had been confirmed luckily against the native proteins with other immunodetecting methods. So working on conditions with Western blot is the first thing to try. 

We suggest you try some experimental conditions, including enriching the proteins loaded on the gel, purifying the native protein from expressed cells, trying reducing conditions, lower antibody concentration, lower incubating temperature (4 oC or 14 oC), higher detergent concentration, blocking the membrane with a higher concentration of BSA or blocking the membrane with donkey and horse serum, and using antibodies from other companies or species. 

We will give you some extra time to decide the fate of your rabbits free of charge, so we will keep rabbits alive and wait for your further tests in your application for the antibodies. Please keep us informed with your results.

Below is our ELISA protocol. Please keep this in mind when using the correct second antibody to do your ELISA tests. 

ELISA Protocol 

Materials: 

1. Solid-phase immobilized Ag: Microtiter wells coated with 1 ug/100 ul/well of peptide BSA are used to capture antibodies. 

2.Antiserum 

3. HRP-conjugated goat anti-rabbit IgG (Pierce). 

4. Diluent for antibody or enzyme conjugate: 2% BSA in PBS. 

5.DI water. 

6.ABTS (Pierce) 

Methods: 

1. Add 100 ul of 10-fold serially diluted antibody to each well and incubate for 30 minutes at 37 oC. 

2.Decant.  Wash three times with excessive DI water. 

3. Add 100 ul of HRP-conjugated goat anti-rabbit IgG (1:4,000) to each well and incubate for 30 minutes at 37 oC. 

4.Decant.  Wash three times with excessive DI water. 

5. Add 100 ul of ABTS substrate solution to each and incubate at room temperature for 20 minutes. 

6. Read plate A405. 

Microtiter well strip setup: 

Dilutions of pre-immune serum: 1:1K dilution 1:10K dilution 1:100K dilution 

Dilutions of antiserum:              1:1K dilution 1:10K dilution 1:100K dilution

Usually, the titers of the antibody will keep rising from day 10 for 6–8 weeks since the 4th immunization. The reboot of the animal should increase the titers of the antiserum at this moment; however, it may also increase the background in some cases. 

Actually, it's very unpredictable if a designed peptide antibody is against the native protein. The reason is that native proteins may be quite different from peptides in many ways, like three-dimensional folded structure, glycolysis, and surface lipids, which could all mask the epitope. Please bear in mind that the immune system is only responding to the epitope, not the sequence molecule. So, in our industry, we only guarantee the antibody against the peptide but not the native protein. Besides, based on our experiences, some antibodies did not work in immunohischemistry, even though the antibodies had been confirmed to be against the native proteins with other immunodetecting methods. We know somebody who can solve this problem by mixing antigens and immunizing the animals. For example, with a mixture of two peptides, one comes from the N-terminal and another from the C-terminal of the same protein. The affinity purification of the crude serum also improves the properties of the antibody by relatively increasing the concentration of specific IgGs.

Some other clues to consider:

We would like to suggest you change some experimental conditions first, including lower antibody concentration, lower incubating temperature (4 oC or 14 oC), higher detergent concentration, blocking the membrane with a higher concentration of BSA or blocking the membrane with donkey and horse serum, and the second antibody from other companies or species.

You may try to affinity purify the antiserum using a column coupled with specific peptides. In our experience, most customers were very pleased with their affinity-purified antibodies because the purified antibody could remove the background effectively. However, a few customers were not so lucky, especially since the proteins came from plants or bacteria.

If your protein is from plants or bacteria, you may try the different eluting fractions from the affinity column. This method works well for some of our customers. Another option is to make polyclonal antibodies from chicken; this will remove the cross-reactions between mammals.

We offer affinity purification to our customers. This service includes coupling 10 mg of free peptide to the affinity resin (5 ml), purifying 50–100 ml of the antiserum, and providing around 5–10 mg of purified antibody together with the ELISA data on every stage of the purification. The price is $500 for 50 ml of antiserum purification and $600 for 100 ml of antiserum. Please let me know if you have additional questions.

Boosting the immunization of rabbits will help increase the antibody titer of antisera. So that's why we provide the option to our clients. We can process the re-boosting option for you right away if you are interested in it. Please let me know your decision at your earliest convenience.

Yes, we have the affinity-purification service available. The price is $600 for the purification of 100 ml of antiserum and $500 for 50 ml, which includes 5 ml of affinity-gel conjugated 10mg of the peptide, purified IgG, and ELISA data on every stage of the purification, and the used antiserum will be returned to you together. In most cases, the yield will be 5–10 mg of purified antibody from 50 ml of antiserum, and the affinity gel can be used multiple times for several years.

It's very unpredictable if a designed peptide antibody is used against native proteins. Most customers designed at least two peptides to generate antibodies against the same protein. We guarantee that the antibodies should be against the peptides. So, please do the ELISA test with the peptide; if the titers of the antibodies are very low against the peptides, the antibodies are bad. We should redo them for free after we confirm your results by ELISA.

It's very hard to identify the specific bands with too much background in Western blots. I suggest that you test the antibodies with immunoprecipitation (IP). Don't worry about the background, as long as the target bands come out. Most backgrounds can be removed by the affinity purification of the crude antiserum, and the purified IgG can be used in most immunodetecting experiments. So, a higher concentration of the antiserum is recommended in the IP (1:25, 1:50, or 1:100).

The idea is that the target protein is first pulled down by the IgG in the antiserum, and then the target proteins can be probed by the anti-tag antibody or the antiserum itself. This experiment can remove most backgrounds. The outline of the protocol is listed below. If the protein is cloned from the expression of the constructs, I would strongly recommend you use the monoclonal anti-tag antibody as the primary antibody to do the Western blot; the results will be much better than the antiserum.

1. Harvest the cells, make the cell extracts (1 ml of sample buffer for each 10cm dish), and collect the lysate in a 1.5 ml eppendoff;

2. Add 50 ul of antiserum to 1 ml of the lysate, vortex, and slowly rotate the eppendoff from end to end at 4 oC for at least 3 hours.

3. Add 25–50 ul of Protein A or G agrose beads to the tube and continue the rotation for overnight at 4 oC;

4. Spin down the beads and draw off the supernant with a 1 ml syringe with a blend needle.

5. Resuspend the beads in 500–1000 ul of 1 X TTBS buffer, briefly vortex and spin down the beads, and discard the supernant;

6. Repeat the wash at least four times.

7. Add 30–50 ul of protein loading buffer to the beads.

8. Mix well by vortexing and heat the sample for 5–10 minutes at 90–100 oC, then put the tub on the ice.

9. Briefly spin down the sample and load 10–20 ul of sample onto SDS-PAGE to do the Western Blots.

To make a good antibody, some researchers try to generate the antibodies at different sites on the protein sequence and ideally select three sites—the N-terminal, C-terminal, and middle—to do the immunization. Believe it or not, the different sites of the peptide will give you different results. So, it's not surprising that the author used different antibodies to do the different experiments. Another option is to make two peptides and mix them to immunize the same animal.

Actually, both human and rat sequences are good for antibody production.

The antibody raised by one of both sequences will recognize both.

peptides. Please bear in mind that the immune system is only responding to the epitope, not the sequence molecule. Sometimes, the antibody may be only against the peptide but not the native protein; that's because the peptide forms a different epitope after folding. So, in our industry, we only guarantee the antibody against the peptide but not the native protein.

The human sequence antibody works in human tissue, so it's for sure the antibody against native protein. In most cases, if the antibody is against a human protein, it will also be against the same protein in other species. This is an advantage of the polyclonal antibody. I had been using a polyclonal antibody from the rat sequence to do lots of experiments with human and mouse tissues.

Here is the protocol for our KLH-peptide conjugation. Briefly, we weigh 15 mg.

KLH reacts with the SMCC linker in PBS. After the PD-10 column was removed, it was unbound.

SMCC and 15mg of free peptide were added to the SMCC-linked KLH for coupling.

overnight. Finally, the free peptides were removed by exhausting dialysis.

The yield of affinity purification is very diverse in different rabbits and depends on the immune response of the individual animal to the antigen. In most cases, we obtained 5 to 10 mg of 96-99% purified IgG from 50ml antiserum. The ELISA range of the purified antibodies is 1.5–3.0 with a 1:1k dilution of 1mg/ml; over 80% of antibodies have ELISA titers above 2.0. The efficiency is almost 100% for the conjugation of Biotin to IgG.

We used to cut a piece of the yolk and weight it, then add the DD water into the tube, break the yolk with a glass bar or the pipette, and vortex. Also, you may bring the yolk back by putting the frozen tube into the water bath at 37–50 oC and transferring the viscous yolk with a blade. The yolk is always viscous; you should weigh it and dilute it by the w/v.

The anti-chicken second antibodies are available in most companies. It's from the MolecularProbe company, which I used for the Cell staining and Western blot.

The simplest treatment is to add 10 folds of dd water at pH 5.2 to the egg yolk, incubate for 30–60 minutes at RT, and then centrifuge for 10–20 minutes at the top speed of your eppendorf. The supernatant can be used for Western and ELISA after further dilution. We can send more information about the IgY purification if need be.

We use the tip to remove and stir the stock yolk and mix it with the water well. The 37-oC water bath will be helpful. If there is still something on the wall, don't worry about it; we don't have to get them all down, or you can let the tube stand longer and go ahead to do the centrifuge. Also, you can incubate the tube at 4 oC overnight by slowly rotating.

In our company, the injection amount of antigen is 25–50 micrograms, with the smallest volume possible for each immunization per mouse based on the KLH concentration. For rabbits, the injection amount is 0.5 mg/0.5 ml at multiple sites per rabbit. Usually, we used to do four immunizations for each project and test the bleed after the third immunization, and this schedule worked well for most projects. We don't care about the allergic reactions in our industry.

We test the immune response of animals to the antigen after the third injection, first by ELISA and then by Western Blot or IP. Unlike your protocols, we do the ELISA with a different carrier protein-conjugated peptide from the immunogen. For example, if the animals are immunized with KLH conjugations, BSA conjugations should be used for the ELISA. Usually, the ELISA data indicate the immune response of the animal to the peptide, and Western Blots and IP will tell us if the antiserum recognizes the native protein. The weakness of the ELISA with the same conjugated peptide is that this kind of data only indicates the general response to the whole thing of the antigen, not the specific peptide and the native protein.

We used to coat 1 ug of the conjugated peptide per well for the ELISA test.

and the range is 1–100 ug per well in the industry. However, the range will

be 0.1–1 ug in most academic institutes.