General Troubleshooting Guide for Antibody

The rules you should pay attention to are as follows: the species of samples, the host species of antibodies, the labeling of antibodies, the recommended dilution ratio for the appliaction of interest. CUSABIO guarantees its antibodies work for the applications and react with the species that listed on the website or product data sheet. If you're working with an application that we did not test, we will offer you a trial size sample to evaluate the antibody before purchasing full size.

Q: What is the difference between monoclonal antibodies and polyclonal antibodies?

Monoclonal antibodies are made using identical immune cells that are all clones of a specific parent cell. As such, they will have affinity for the same antigen and epitope.Polyclonal antibodies are made using several different immune cells. They will have affinity for the same antigen but different epitopes.Monoclonal antibodies bind only a single epitope while polyclonal antibodies bind different epitopes on the same protein. Monoclonal antibodies are much more specific and with less background noise than polyclonal antibodies, and are as such generally preferable for biochemical assays. However, they are also usually more expensive to produce, less robust and are very sensitive to small changes in the antigen.

Q: What is the relationship between primary antibody and secondary antibody? How can I select the right secondary antibody for my experiment?

In general, primary antibody is used to capture the target protein, secondary antibody can bind to primary antibody and is labeled with a enzyme or dye that can be amplified for detection. We suggest you consider the following elements:

a. Depending on the host species. For example, if the primary antibody is from mouse, the secondary antibody should be anti-mouse.

b. Depending on the characteristics of primary antibody.

c. Depending on your applications, such as WB, IHC, ELISA, etc.

Q: Can you please let me know how many biotins per antibody are bound?

The molar ratio of biotin to antibody is 36.6:1. However, it's difficult to say exactly how many biotins are conjugated per antibody, as labeling is via the lysine residue and it's not clear how may primary amine groups are attached to biotin. On average, 3-5 biotin molecules can be conjugated per IgG molecule.

Q: Why is the actual band greater than the theoretical molecular weight?

You can calculate the antibody molecular weight using CUSABIO Molecular Weight Calculator. If there is a big difference between the test results and the theoretical molecular weight. The possible causes are as follows: The target protein has multiple modification sites such as glycosylation, or it forms stable complexes with other proteins in the body, or it is cut or degraded.

Q: Why is the volume of this antibody I received less than that stated on the vial label?

Small volumes of antibody may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container's cap.

Q: Can you recommend isotype control for the antibody?

Isotype controls are used to confirm that the primary antibody binding is specific and not a result of non-specific Fc receptor binding or other protein interactions. The isotype control antibody should match the primary antibody’s host species, isotype and conjugation. For example, if the primary antibody is a FITC-conjugated mouse IgG1, then you will need to choose a FITC-conjugated mouse IgG1 isotype control. Most isotype controls are monoclonal. Polyclonal antibodies are not suitable as these antibodies contain more than one IgG subclass.

Q: Which buffer is used to store antibodies?

We use 0.01M PBS (pH 7.4) with 50% glycerol.

Q: What kind of antibody labels can you offer?

We currently provide FITC, HRP, Biotin random labeling at the free lysine (lys) amino groups.

Q: Why are some antibodies no longer available ?

We remove these antibodies from our stock because we have new products replacement.

Q: What is a certain antibody?

Antibody (Ab), also known as immunoglobulin (Ig), is a Y-shaped protein that is used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses via the Fab's variable region.

Q: Why are there no bands in western blot (WB)?

a. Lacking of antibody. Some antibodies may have poor affinity to target proteins. You should reduce antibody dilution (2-4 fold lower than recommended starting dilution). Meanwhile, antibody may have lost activity. You should perform a dot blot assay.

b. Lacking of protein. You should increase the amount of total protein loaded on gel. Confirm the presence of protein by another method. Use a positive control (recombinant protein, cell line or treat cells to express analyte of interest). Perform a dot blot assay.

c. Poor transfer. You should wet PVDF/Immobilon-P membrane in methanol or nitrocellulose membrane in transfer buffer to ensure good contact between PVDF membrane and gel.

d. Incomplete transfer. You should extend transfer time, because some proteins with high molecular weight may require some more time on transfer.

e. Over transfer. You should reduce transfer voltage or time for those proteins with small molecular weight (lower than 10 kDa).

f. Isoelectric point is bigger than 9. You should use alternative buffer system with higher pH value, such as CAPS (pH 10.5).

g. Incorrect secondary antibody used. You should confirm host species and antibody type of primary antibody to choose the right secondary antibody.

h. Antibody doesn't work. Don’t use the antibody beyond the expiration date.

i. Sodium azide contamination. You should make sure buffers do not contain Sodium azide for it can quench HRP signal.

j. Insufficient incubation time for primary antibody. You should extend incubation time to overnight at 4℃.

Q: Why is the signal weak in western blot (WB)?

a. Low protein-antibody binding. You should minimize the number of washes. Reduce NaCl concentration in antibody solution or use blotting buffer for wash steps (recommended range 0.15 M-0.5 M).

b. Insufficient antibody. You should increase antibody concentration to 2-4 fold higher than recommended starting concentration.

c. Insufficient protein. You should increase the amount of total protein loaded on gel.

d. Inactive conjugation. You should mix enzyme and substrate in a tube. If color does not develop or, it is weak, make fresh or purchase new reagents. Switch to ECL.

e. Weak/Old ECL. You should purchase new ECL reagents.

f. Non-fat dry milk may mask some antigen. You should decrease percentage of blocking milk or substitute with 3% BSA.

Q: Why are there extra bands in western blot (WB)?

a. Non-specific binding of primary antibody. You should increase primary antibody dilution factor. Reduce the amount of total protein loaded on gel. Use mono-specific or antigen affinity purified antibodies.

b. Non-specific binding of secondary antibody. You should run a control with the secondary antibody alone (omit primary antibody). If bands develop, then choose an alternative secondary antibody, use mono-specific or antigen affinity purified antibodies.

c. Degradation of analyte. You should minimize freeze/thaw cycles of sample, add protease inhibitors to sample before storage or make fresh samples.

d. Aggregation of analyte. You should increase amount of DTT to ensure reducing the disulfide bonds (20-100 mM DTT). Heat in boiling water for 5-10 minutes before loading onto gel. Perform a short-time centrifugation.

e. Contamination of reagents. You should check whether the buffers had been contaminated or not, or make fresh reagents alternatively.

Q: What is the reason for a smear in a western blot?

a. Non-specific binding of primary antibody. You should dilute mono-specific or antigen affinity-purified antibodies in 5% milk at a lower concentration or adjust the non-fat milk (2-5%) or NaCl (0.15-0.5 M) concentrations as primary antibody solution.

b. Non-specific binding of secondary antibody. You should run a control with the secondary antibody alone (omit primary antibody). If there are some bands appearing on the membrane, you should dilute the antibody or change another secondary antibody alternatively.

c. Insufficient blocking. You should start with 5% non-fat dry milk with 0.1%-0.5% Tween 20, 0.15-0.5 M NaCl in 25 mM Tris (pH 7.4). Incubation time could be extended. Adjust milk concentration up or down based on needs. Overnight blocking at 4℃ may decrease blocking efficiency since detergents might not be effective at lower temperatures.

d. Insufficient wash. You should increase the concentration of Tween 20 in wash buffer (0.1%-0.5%) or increase the number of washes.

e. Non-fat dry milk may contain target antigen or endogenous biotin and is incompatible with avidin/streptavidin. You should substitute with 3% BSA.

f. Some IgY antibodies may recognize milk protein. You should substitute with 3% BSA.

g. Film overexposed. You should reduce exposure time. If target signal is too strong, then please wait 5-10 minutes more and re-expose to film.

Q: Why does the band look diffuse and irregular in western blot (WB)?

a. Bad gelling. You should mix the solution thoroughly before filling.

b. Some samples have higher salt concentrations. You should desalt or adjust the salt concentration of the sample.

c. Inconsistent sample volume per well. You should adjust the sample volume so that it is basically the same.

d. Buffer doesn’t work. You should purchase a new buffer or make fresh buffer.

e. Air bubble trapped in membrane. You should gently remove any air bubbles especially during transfer.

f. Excessive temperature during electrophoresis. You should reduce current or voltage.

Q: Why is transfer efficiency low in western blot (WB)?

a. PH is close to the isoelectric point. You should increase the pH of the transfer buffer.

b. Air bubble trapped in membrane. You should gently remove any air bubbles. Especially during transfer.

c. Filter paper contact. The size of filter paper, transfer film and gel should be basically the same to avoid direct contact of filter paper.

d. Improper selection of transfer film. You should use reliable PVDF film or nitrocellulose film.

e. Too low voltage or current. We recommend 20 mA constant current during wet transfer, 25 V constant voltage around semi-dry transfer.

f. Too long or too short transfer time. You should select the appropriate transfer time according to protein size and transfer device using electrophoresis,.

g. Excessive ambient temperature during wet transfer. You should use pre-cooled transfer buffer or set the unit to 4℃.

Q: Why is staining lack in immunohistochemistry (IHC)?

a. Lack of antigen. We suggest to examine the protein expression via in situ hybridization, because in some rare situations, the protein translation may be blocked even though mRNA had been detected.

b. Antigen was destroyed before incubation with the primary antibody. You should block peroxidase after incubation with the primary antibody if quenching of endogenous peroxidase was done prior to the addition of primary antibodies.

c. Antigen retrieval was ineffective. You should increase the time of treatment or change the treatment solution.

d. Antibodies do not work due to improper storage. You should follow storage instructions on the manual. In general, aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Please avoid repeated freeze-thaw cycles.

e. Inactive primary or secondary antibodies. You should test reporter system independently to assess reagent viability.

f. Incompatible secondary and primary antibodies. You should use a secondary antibody that will interact with the primary antibody. For example, if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody.

g. Inadequate tissue fixation. You should try increasing the fixation time or try a different fixative.

h. Tissue overfixation. You should reduce the duration of the immersion or post-fixation steps. If immersion fixation cannot be avoided, antigens may be unmasked by treatment with antigen retrieval reagents.

i. Epitope altered during fixation or embedding procedure. You should try restoring immunoreactivity through various antigen retrieval techniques.

j. Reagents omitted or used in wrong order. You should repeat staining and confirm that correct reagents are used in the correct order.

Q: Why is staining inappropriate in immunohistochemistry (IHC)?

a. Fixation method is inappropriate for the antigen. You should try a different fixative or increase the fixation time.

b. Antigen retrieval may be inappropriate for this antigen or tissue. You should try different antigen retrieval conditions.

c. Electrostatic charge of the antigen has been altered. You should try adjusting the pH or cation concentration of the antibody diluent.

d. Delay in fixation caused diffusion of the antigen. You should fix tissue promptly and try a cross-linking fixative rather than organic (alcohol) fixative.

Q: Why is background high in immunohistochemistry (IHC)?

a. High concentration of primary and or secondary antibodies. You should titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies.

b. Non-specific binding of primary or secondary reagents to tissues. You should use blocking step just prior to primary antibody incubation. Non-fat dry milk is another option.

c. Non-specific binding of secondary antibody. You should use an antibody that has cross-reactive IgG species removed (absorbed against sample species).

d. Hydrophobic interactions of the antibody and proteins in the tissue. You should lower the ionic strength of the antibody diluent.

e. Background due to ionic interactions. You should increase the ionic strength of the dilution buffer.

f. Tissue dried out. You should avoid letting the tissue dry during the staining procedure.

g. Reagents sticking to old or poorly prepared slides. You should start over with freshly prepared or purchased slides.

Q: Why is tissue or cell morphology destroyed in immunohistochemistry (IHC)?

a. Antigen retrieval methods are too harsh. You should empirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen.

b. Tissue sections falling off slide. You should increase fixation time. Empirically determine an additional or alternative fixative. Use freshly prepared, adequately charged slides.

c. Underfixation has physically damaged the tissue or cells during the staining process. You should increase fixation time and/or add a post-fixation step. Increase the fixative/tissue ratio. Cut smaller pieces of tissue for more thorough immersion fixation.

d. Autolysis of tissue leading to staining of necrotic debris. You should increase the fixation time, ratio. Consider using cross-linking fixative.

e. Tissue section appears torn or folded. You should remove air bubbles under section. Re-cut sections using a sharp blade, or ignore damaged areas when analyzing the results.

f. Poor resolution of tissue morphology. You should cut thinner tissue sections as ice crystals may have destroyed morphology of frozen sections.

Q: Why is there too much antibody eluting in immunoprecipitation (IP)?

You should mix the antibody with the beads before IP and use a gentle glycine buffer gradient to elute that will significantly reduce the amount of antibody.

Q: Why is the background so high in immunoprecipitation (IP)?

a. Too much non-specific binding to antibody. You should use the purified antibody in a less amount.

b. Too many cells/too much protein in lysate leading to a lot of non-specific proteins in eluate. Reduce the number of cells/lysate used (10-500 µg).

c. Carry over of proteins that are not detergent soluble. Remove supernatant immediately after centrifugations. This should leave insoluble proteins in the pellet. If resuspension occurs, centrifuge again.

d. Too less prepare beads. You should make sure BSA is fresh and incubate enough beads for 1 hour with 1% BSA in PBS. Wash 3-4 times in PBS before using them.

e. Incomplete washing. You should wash thoroughly at relevant stages by placing a lid on the tube and inverting several times before centrifuging.

f. Antigen degrading during immunoprecipitation. You should add fresh protease inhibitors before lysating.

Q: Why is low resolution with high background across large regions in chromatin immunoprecipitation (ChIP)?

You should optimize the DNA fragment (no larger than 1.5 kb).

Q: Why is there no DNA amplification from samples in chromatin immunoprecipitation (ChIP)?

You should choose positive control template DNA to confirm that the primers are working well.

Q: Why is high background in flow cytometry (FC)?

a. Too high gain set. You should reduce the gain to decrease the signal and use the positive control to set up the flow cytometer correctly again.

b. Too much antibody. You should decrease the antibody concentration.

Q: Why are there two or more cell populations observed in flow cytometry (FC)?

a. More than one cell population expressing target protein. You should check if there is adequate cell separation.

b. Cell doublets present. Doublets of cells will show as a second cell population at approximately twice the fluorescence intensity on the plot. You should mix cells gently using a pipette before staining and before running on the cytometer.

Q: How should I store and aliquot the antibody?

The antibody is stable at room temperature for only a few days. Please aliquot and store the antibody at -20℃ upon delivery. Please avoid repeated freeze and thawing that you can aliquot antibodies according to the amount used in your experiment, but each aliquot should not be less than 10 μL. The less amount of each aliquot, the greater impact is on antibody concentration due to liquid evaporation and container adsorption.