Frequently Asked Questions
What is the procedures for phospho-peptide purification? How it works? (Top)
We applied the antiserum from rabbit to 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 client). The eluted fraction of the non-phosphopeptide column which will mark with anti-non-phosphopeptide, but please be aware, those fraction do not only bind to non-phosphopeptide, but also bind to phosphopeptide. That's why it reacted preferentially with the phospho-form because it's 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 non-phosphopeptide column did only bind to the phosphopeptide, so it's real anti-phosphopeptide antibody. In our experience, it's better to re-purify this fraction by the phosphopeptide column to get real specific anti-phosphopeptide antibody. The eluted fractions of the re-purification with phosphopeptide column are the final purified antibody. These two should be exactly same.
To get the real anti-non-phosphopeptide antibody, the antiserum must be applied to the non-phosphopeptide column first, and then remove the non-specific IgG by phosphopeptide column. We can do this purification with the used antiserum. The price is $700.
Does the phospho-antibody work with phosphorylated protein? (Top)
We did provide you with antibody ELISA test against phospho-peptide and non-phospho peptide, it will be upon to you to test with corresponding phospho- proteins 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 big gap between peptide and whole protein due to conformation and other structure difference, for example glycolysation. But we have had experience that some of our client's 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 first thing to try.
We would like to suggest you to 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 (4oC or 14oC), higher detergent concentration, blocking the membrane with higher concentration of BSA or blocking the membrane with donkey and horse serum, and the second antibody 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
What is your ELISA protocol? (Top)
Below is our ELISA protocol. Please bear mind, using the correct second antibodies to do your ELISA tests.
1.Solid phase immobilized Ag: Microtiter wells coated with 1ug/100ul/well of
peptide BSA are used to capture antibody.
3.HRP-conjugated goat anti-rabbit IgG (Pierce).
4.Diluent for antibody or enzyme conjugate: 2% BSA in PBS.
1.Add 100 ul of 10-fold serially diluted antibody to each well and incubated for 30 minutes at
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 37oC.
4.Decant. Wash three times with excessive DI water.
5.Add 100ul of ABTS substrate solution to each and incubate at room temperature for 20
6.Read plate at 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
How long the titer will remain after last booster? How to reduce the background and to have a better chance to see reaction with native protein? (Top)
Usually, the titers of the antibody will keep at higher level from day 10 for 6-8 weeks since the 4th immunization. The reboot on the animal should increase the titers of the antiserum at this moment, however, it may be also increase the backgrounds in some cases.
Actually, it's very unpredictable, if a designed peptide antibody is against the native protein. The reason is that native protein may be quite different from peptides in many ways like three dimesional folded structure, glycolysation and surface lipids, which all could mask the epitope. Please bear mind, the immune system is only response to the epitope, but not the sequence molecule only. 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 never work in immunohischemistry, even the antibodies had been confirmed really against the native proteins with other immunodetecting methods. We know somebody to solve this problem by the mix antigens immunization of the animals. For example, with the mixture of two peptides, one is come from the N-terminal and another one C-terminal in same protein. The affinity-purification of the crude serum also improves the property of the antibody with relatively increasing the concentration of specific IgGs.
Some other clues to consider:
We would like to suggest you to change some experimental conditions first, including lower antibody concentration, lower incubating temperature (4oC or 14oC), higher detergent concentration, blocking the membrane with 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 by the column coupled with specific peptides. In our experience, most customers were very please with their affinity-purified antibodies, because the purified antibody could remove the background effectively. However, few customers were not so lucky, especially for the proteins came from plants or bacterial.
If your protein is from plants or bacterial, you may try the different eluting fractions from affinity column. This method works well in some our customers. Another option is to make polyclonal antibody from chicken, it will remove the cross reactions between the mammalian.
We offer the affinity purification to our customers. This service includes coupling of 10 mg of free peptide to the affinity-resin (5ml), purifying 50-100ml of the antiserum, providing around 5-10mg of purified antibody together with the ELISA data on the every stage of the purification. The price is $500 for 50ml of antiserum purification, and $600 for 100ml of antiserum. Please let me know, if you have additional questions.
Boosting immunization of rabbits will help to 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. Please let me know your decision at your earliest convenience.
Do you do affinity purification? (Top)
Yes, we have the affinity-purification service available. The price is $600 for purification of 100ml antiserum and $500 for 50ml, which includes 5ml 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 return to you together. The yield will be 5-10mg purified antibody from 50ml of antiserum in most cases, and the affinity-gel can be used multiple times for several years.
What is the chance that your customer-designed antibody can recognize native protein? How to get a single band in Western Blot test with your antibody? (Top)
It's very unpredictable if a designed peptide antibody against native proteins. Most customers designed at least two peptides to generate the antibodies against the same protein. We guarantee 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 confirmed your results by ELISA.
It's very hard to identify the specific bands with too much background in Western blots. I would like to suggest you to test the antibodies with the immunoprecipitation (IP). Don't worry about the background, as long as the target bands coming out. The 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, 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 firstly 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 listed below. If the protein is cloned from the expression of the constructs, I would strongly recommend you to 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 and make the cell extracts (1ml of sample buffer for each 10cm dish), and collect the lysate to 1.5ml eppendoff;
2. Add 50ul of antiserum to 1ml of the lysate, vortex and slowly rotate the eppendoff by end to end at 4oC for at least 3 hours;
3. Add 25-50ul of Protein A or G agrose beads to the tube, and continue the rotating for overnight at 4oC;
4. Spin down the beads, and draw off the supernant by a 1ml syringe with a blend needle;
5. Resuspend the beads in 500-1000ul of 1 X TTBS bffer, briefly vortex and spin down the beads, and discard the supernant;
6. Repeat the wash at least 4 times,
7. Add 30-50ul of protein loading buffer to the beads;
8. Mix well by vortexing and heat the sample for 5-10 min at 90-100oC and then put the tub on the ice;
9. Briefly spin down the sample and the load the 10-20ul of sample onto SDS-PAGE to do the Western Blots.
How to make a good antibody? (Top)
To make a good antibody, some researchers try to generate the antibodies by different sites on the protein sequence, and ideally select 3 sites, N-terminal, C-terminal and the middle, to do the immunization. Believe or not, the different site peptide will give you different results. So, it's not surprise for that author used different antibodies to do the different experiments. Another option is that you may make two peptides and mix them to immunize the same animal.
Actually, both human and rat sequences are good for the antibody production.
The antibody raised by one of both sequences will recognized both
peptides. Please bear mind, the immune system is only response to the epitope, but not the sequence molecule only. Sometimes, the antibody may be only against the peptide but not the native protein, that's because the peptide form a different epitope after the 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 case, if the antibody is against human protein, it will be also against same protein in other species. This is an advantage of the polyclonal antibody. I had been used a polyclonal antibody from rat sequence to do lots of experiments with human and mouse tissues.
In respect to use the conjugates to immunize mice for the following hybridoma production, most people used to use 50 ug of a purified antigen per injection per mouse. Because the antigen concentration in these preparations is unclear, how much the KLH from the conjugate's tubes should be used per injection per one mouse? How many injections do you expect to induce specific response? Do you predict allergic reactions due to big amounts of KLH injected? (Top)
Please, specify the concentration of conjugates that will produce specific signal in comparison to pure KLH in ELISA?
In our company, the injection amount of antigen is 25-50 microgram with smallest volume possible for each immunization per mouse based on the KLH concentration. For rabbit, the injection amount is 0.5 mg/0.5 ml multiple sites / per rabbit. Usually, we used to do 4 times of immunization for each project, test the bleed after the third immunization, and this schedule works well in most projects. We don't care about the allergic reactions in our industry.
We test the immune response of animal to the antigen after the third injection, firstly by ELISA and then by Western Blot or IP. Unlike your protocols, we do the ELISA with different carrier protein conjugated peptide from the immunogen. For example, if the animals are immunized by KLH conjugations, the BSA conjugations should be used for the ELISA. Usually, the ELISA data indicate the immune response of the animal to the peptide, Western Blots and IP will tell us if the antiserum recognizes the native protein. The weakness of the ELISA with same conjugated peptide is that this kind of data only indicates the general response to the whole thing of the antigen, but not the specific peptide and the native protein.
We used to coat 1ug of the conjugated peptide per well for the ELISA test,
and the range is 1-100ug per well in the industry. However, the range will
be 0.1-1ug in most academic institutes.
Please clarify the KLH-conjugation procedures (Top)
Here is the protocol of our KLH-peptide conjugation. Briefly, we weight 15mg
KLH reacting with SMCC linker in PBS. After PD-10 column removed unbound
SMCC, 15mg of free peptide were added to the SMCC linked KLH for coupling
overnight. Finally, the free peptides were removed by exhaustedly dialysis.
How much antibody we will expect to get after affinity purification? (Top)
The yield of affinity-purification is very diversity in different rabbit and is depending on the immune response of the individual animal to the antigen. We got 5 to 10 mg of 96-99% purified IgG from 50ml antiserum in most case. The ELISA range of the purified antibodies is 1.5-3.0 with 1:1k dilution from 1mg/ml, over 80% of antibodies is above 2.0 ELISA titers. The efficiency is almost 100% for the conjugation of Biotin to IgG.
Chicken York is frozen, too viscous. Do you have a better way to defrost? (Top)
We used to cut a piece of the yolk and weight it, then add the dd water into the tube, break the yolk by a glass bar or the pipette and vortex. Also, you may bring the yolk back by put the frozen tube into the water bath at 37-50oC, transfer the viscous yolk by a blade. The yolk is always viscous, you should weight it, and dilute it by the w/v.
How to purify or simple test of Western Blot. Which company you have order HRP-conjugated anti-Chicken antibody? (Top)
The anti-chicken second antibodies are available in most company. It's from MolecularProbe company which I used for the Cell staining and Western blot.
The simplest treatment is adding 10 folds of dd water at pH5.2 to the egg yolk, incubating for 30-60 minutes at RT, and then centrifuge for 10-20 min at top speed of your eppendorf. The supernant can be used to do western and ELISA after further dilution. We can send more information about the IgY purification if need be.
What did you do when egg york stuck to the side of tubes? some of them make good mix, other just stuck to the wall. (Top)
We use the tip to remove and stir the stock yolk and mix it with water well. The 37oC 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 standing longer, and go ahead to do the centrifuge. Also, you can incubate the tube at 4oC overnight by slowly rotating
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