FAQ about Agrisera IncuBlocker

Method questions

Q: What makes detection using IncuBlocker faster than alternatives?
A: Using Agrisera IncuBlocker, all steps e.g. blocking, primary and secondary antibody incubations are carried out at the same time. This reagent is optimized for efficient membrane blocking following the transfer and antibody incubations, all at once. Agrisera IncuBlocker is blocking the membrane much faster and more efficiently what eliminates non-specific background and makes it more difficult for primary and secondary antibodies to bind to the membrane non-specifically. Such non-specific background has to be prevented by washing and incubation steps using standard western blot protocol. This way Agrisera IncuBlocker can considerably save time compare to standard western blot protocol by shortening western blot procedure by several hours.

Q: Can I re-use the Agrisera IncuBlocker solution which contains my primary antibody?
A: Yes, it is going to work for antibodies of high and medium titer which are binding to proteins of high or medium abundance in your sample. Usually this solution can be re-used up to 5 times. With every use, amount of primary antibody is decreased and bands may become weaker. This can be compensated by increasing exposure time on X-ray film or images. During more excessive use, both, blocking reagent and secondary antibody will be consumed contributing to higher background and weaker bands. This approach is not recommended for quantitative western blot.

Q: What Agrisera IncuBlocker volume should be used to incubate my membrane?
A: All volumes listed in instructions  apply to the dimensions of the membrane for a minigel (10 x 8 cm). If membranes of others sizes are used, volume needs to be adjusted. In case of vessels or tubes of very low volume, do not reduce Agrisera IncuBlocker volume significantly as this can contribute to background problems.

Q: Which detection methods are compatible with Agrisera IncuBlocker?
A: Agrisera IncuBlocker (rabbit) contains HRP-conjugated goat anti-rabbit secondary antibodies and can be used with any ECL substrates visualized by X-ray films or imagers. For detection using TMB substrate larger amount of protein should be loaded per well. Agrisera offers IncuBlocker solutions for other species as well including: chicken, mouse and goat.

Q: What equipment and chemicals do I need to use Agrisera IncuBlocker?
A: The gel running and the blotting steps are unchanged. Following blotting, whole procedure is simplified and shortened by using accelerated blocking and incubation with primary and secondary antibody (included already in IncuBlocker). Final visualization step can be performed using AgriseraECL BrightEnhanced.

Q: Which membrane can I use with Agrisera IncuBlocker?
A: This reagent is optimized to work with most common membranes including PVDF and nitrocellulose as well as membranes used in "Turbo" or Fast blotters.

Q: What does Agrisera IncuBlocker kit contain?
A: This kit includes stock solution of Wash Buffer (solution A) and detection solution with appropriate secondary antibody, which is in this case goat anti-rabbit (solution B). Stock solution of Wash Buffer can be also purchased separately as well as AgriseraECL BrightEnhanced.

Results questions

Q: Can results obtained with Agrisera IncuBlocker be worse due to a rapid blocking and binding process?
A: No, if antibodies of high titer are used. Characteristics of your primary antibody are most important as final results will depend upon it.

Q: What to do if I observe background signal with Agrisera IncuBlocker?
A: Increase incubation time to 1-2 hours.

Q: What can happen if I use another washing buffer?
A: Higher background can be obtained. Agrisera IncuBlocker Wash Buffer is optimized for fast all-in-one-detection.

First test with Agrisera IncuBlocker

Q: How to test compatibility of my primary antibody with Agrisera IncuBlocker?
A: Prepare a membrane by transferring your proteins to it with identical samples and cut it into two identical halves. Use your standard protocol on one half while Agrisera IncuBlocker protocol on the other half.



Work with a known primary antibody is recommended. The primary antibody should be used at 0.5 ug/ml if it is affinity purified or at dilution which you usually apply for this specific antibody. Initially develop reaction following 1 hour. This time can be shortened as on example which is shown above. If there is no signal with Agrisera IncuBlocker after 2 hour incubation, your antibody is not suitable for rapid immunodetection with this reagent.

Antibodies

Q: What to do if I do not know specific concentration of my primary antibody?
A: Specific concentration of a primary antibody is known for antibodies which are affinity purified. If your antibody is provided in serum format or as total IgG fraction purified on Protein A or G, follow instruction which is provided on product info sheet for given antibody by its manufacture, e.g. dilution: 1: 5000, for example.

Q: Are all antibodies suitable to work with Agrisera IncuBlocker?
A: Yes, if they are of high titer and good affinity for proteins of moderate or high expression. If your antibody requires over night incubation to visualize the signal, Agrisera IncuBlocker solution is not going to speed up such reaction.

Q: What to do if my primary antibody is made in another species than rabbit?
A: Agrisera IncuBlocker rabbit is prepared to work with primary antibodies made in a rabbit. If your primary antibody is made in another species, like chicken or goat, you need to use AgriseraIncuBlocker for that specific species, e.g. Agrisera IncuBlocker (chicken) or Agrisera IncuBlocker (mouse). For other species, please inquire.

Wash buffer

Q: Why do I have to use specific wash buffer provided together with AgriseraIncuBlocker?
A: Provided wash buffer is optimized for detection together with Agrisera IncuBlocker. Washing step before incubation with Agrisera IncuBlocker is very important and provides least background. Wash buffer can be also purchased separately.

Q: How many washing steps do I need to apply when working with Agrisera IncuBlocker?
A: Washing step allows to remove unbound primary and secondary antibodies to avoid background. It depends upon primary antibody which is used. Usually 3x5 minutes washes are enough, for some antibodies 4-5.