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See below for guidelines concerning proper antibody storage and centrifugation:
• After receiving your antibody，centrifuge for 3 minutes at 12000rpm and 4℃. Remove and open the tube lid for aliquoting and dissolution before storing (if the antibody volume is more than 50uL, extend the centrifugation time to 5 minutes to guarantee the reclamation of all antibodies and recombinant protein).
• For long-term storage of most antibodies, -20℃ is recommended.
• Prolong antibody activity by avoiding freeze / thaw cycles as much as possible. You may also reduce the possibility of contamination caused by taking antibody from same vial several times by avoiding aliquoting.
• To preserve your antibody, aliquoted amounts should be just enough for one experiment and no less than 10ul each. Antibody concentration may be influenced by evaporation and adsorption to the vial wall if the aliquoted amount is less than 10ul.
• If thawed antibody isn’t used up all at once, store the rest of the antibody liquid at 4℃ to avoid refreezing.
• Antibody working liquid should be made up and used in one day. Do not store for more than one day at 4℃.
• Always avoid storing antibodies in an automatic defrost refrigerator/freezer. Automatic defrost cycles cause dramatic changes in the interior temperature of these freezers. If storage in this kind of freezer is absolutely necessary, place the antibody near the center of the refrigerator, because the highest temperature fluctuations occur near the door and walls of the freezer.
• Most antibodies’ activity will not be adversely influenced when stored at 4℃ for 1 - 2 weeks.
• If antibodies are to be used within 1 - 2 weeks, the recommended storage temperature is 4℃. Make sure to avoid freeze/thaw cycles.
• The best long-term storage temperature should be -20℃. Kindly refer to your antibodies’ specifications for proper storage procedures.
• Ascites should be frozen upon reception. Ascites contain proteases which will be degraded if stored at 4℃ for a long period time.
• General shipping can take up to 2 weeks at 4℃ if need be and the antibody should not lose any of it’s effectiveness.
• Transportation with dry ice should be avoided because this will cause another freeze/thaw cycle.
a.For enzyme-linked antibodies:
Always store at 4℃. Avoid freezing，or enzyme activity will decrease.
b.For coupling antibodies：
Must be stored in brown tubes or silver paper, in order to avoid light exposure. Fluorescent antibodies in particular are very sensitive to light, and all experiments involving fluorescent antibodies should be conducted in the dark.
c.For igg3 isotype control antibodies:
Always store at 4℃. Avoid freeze/thaw cycles, which will easily cause the antibody to polymerize.
Specific band confirmation:
Refer to the Predicted MW: most antibody’s detected results are within a 10-15% margin of the Predicted MW. Please refer to the data on the Uniprot website, http://www.uniprot.org/, for the Predicted MW.The possible reasons that the detected MW has differed from the predicted MW:
1.The target protein has modification sites, i.e. a glycosylation.
2.The target protein has combined with another molecule, i.e. another protein.
3.The target protein has been cleaved.
4.There are a few special proteins that are known to show poorly on Western blots such as ubiquitin.
5.Di-polymer or polymer.
LAMP1 has both a 47kDa and 120kDa band. They are both considered specific bands.
After visiting the Uniprot website, http://www.uniprot.org/uniprot/P11279 , the predicted MW is 45kDa.
This target has many glycosylation sites as shown on the Uniprot website.
The detected MW therefore will be larger than the predicted band because of these modifications. You can look at other companies’ testing results for confirmation. We look at ABcam’s and Cell Signaling’s results as examples below.
Our two specific bands were confirmed on numerous other sites so this offers further confirmation of the results.
Please see the testing results of ATG5 for reference. This can be found in the diagram to the right.
The predicted Molecular weight of ATG5 is 32kDa. ATG5 doesn’t undergo glycosylation, however, the ATG5 protein can combine with ATG12 to form a stable protein complex.
According to this information, we can conclude that the MW of the detected band is ATG5+ATG12. We can refer to other companies’ testing results, as shown on the left.Taking these data into consideration, the 55kDa detected band of ATG5 is the specific band.
Please see test result of LTF protein as follow, the predicted MW is 78kDa, but our test result is around 50kDa:
We have questions about the test result and we could not find the relevant data on Uniprot website, however, we found the LTF protein was reported that it could be hydrolyzed into two bands, 50kDa and 30kDa. So the detected band of LTF is the specific band（This protein has 2 isoforms produced by alternative promoter usage. The human LTF can be isolated two fragments， an N-terminal tryptic fragment having an Mr of 30 kDa and a C-terminal tryptic fragment having an Mr of 50 kDa by a mild tryptic digest(PMID:3790094)）。
We use a preservative called thiomersal, NaN3. Many other companies like Abcam and PTG use this preservative as well.
NaN3 functions as a microbe inhibitor.
Those antibodies that contain NaN3 are primarily used for WB, IHC, IF, IP, and FC. Those without preservative are able to be added to certain cell culture experiments. For example, CD3 and CD28 are expected to stimulate the activation of T-lymphocytes.
The NaN3 must not be added to the HRP-secondary antibody. It has an effect on both the activity of the HRP-secondary antibody and will inhibit the coloration.
ABclonal's primary antibodies usually contain trace amounts of NaN3. The NaN3 will usually be washed away in a standard WB experiment before the secondary antibody is loaded so the preservative will not effect the later results.Please Note: review the information of the TBS and PBS to be used as these solutions tend to contain NaN3 and may therefore cause poor results.
To conclude: the NaN3 maintains the stability of the antibody by inhibiting microbial growth. Those antibodies with preservatives must not be used to "study biological function" and/or the “research of living cells”. They must not be used directly with HRP-secondary antibodies either. Other than these specific cases, the NaN3 has been shown to have little to no effect on experiments.
It typically takes between fifteen and seventeen weeks to produce a custom antibody. Our protocol is as follows: Cloning -> Expression -> Immunization -> Affinity Purification -> Testing.
Cloning: We select the expression vector template according to the project report. We sequence the clones to confirm the expression vector. This process takes about two weeks to complete. The customer has the option to provide their own template (15+uL) and sequencing results. This may save some time.
Expression: we use a prokaryotic species to induce expression of our sought-after vector. This will take two weeks.The customer has the option to provide the expression vector and reference chart to save some time.
Immunization: It takes 68 days to run our immunization protocol. Our rabbits undergo four immunizations, with the final round proving to be instrumental in developing top-notch antibodies. The customer has the option to test the anti-serum after the third immunization for their purposes if he/she is under time constraints.
Affinity purification: Our custom antibodies are almost always at a concentration greater than 1.0mg/mL. When diluted 1:1000, the antibody will be able to detect 10ng of antigen. This process will take about three weeks.
Testing: It will take about 3 weeks to test the antibody. And then about 10 days to purify it.If the customer wants, we are able to send the test sample to him/her for endogenous testing. We are able to run antigen testing at the same time.
Our custom antibody is guaranteed to detect the antigen. When the antibody has been developed by the expression protocol, it can be diluted at 1:1000 and can detect 10ng of antigen. When the antibody has been developed using the peptide protocol, it can be diluted at 1:1000 in Dot-Blot and can detect 100ng of antigen.
Note: There are a few antigens that may not be detectable due to inherent properties.
Note: Due to the variability in endogenous testing, we cannot guarantee this result. We will, however, share the risk. If the endogenous testing fails and ABclonal is unable to troubleshoot the problems successfully after reviewing the data and collaborating with the lab members, we will only charge 50%!
After analyzing our more than 5 years of custom antibody project data, the probability of successful endogenous detection using the expression route is 70-80% while for peptide route is 60-70%.