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An immuno-reaction enhancing solution

IMMUNO SHOT Protocol for Western blotting and ELISA

Protocol of Western Blotting (WB)

WB is a method to detect proteins by specific antibodies. Usually, proteins are separated by SDS-PAGE and transferred to membrane made by nitrocellulose or PVDF (polyvinylidene fluoride). The way to use IMMUNO SHOT in WB is described below.

1. SDS-PAGE and transfer of protein to PVDF membrane should be done by usual method.

2. Blocking and the washing should be done by usual method.

3. Dilute the 1st antibody with Reagent 1 of IMMUNO SHOT. The dilution factor is influenced by many factors, such as antibody species, amount of antigen, etc.  Though you can reduce antibody concentration by using IMMUNO SHOT, we recommend performing a pre-test to determine the best antibody concentration.

4. Dilute the 2nd antibody with Reagent 2 of IMMUNO SHOT. The best dilution factor is influenced by many factors, such as antibody species, amount of antigen, etc.  Refer to the supplier’s instruction to determine the best antibody concentration.

5. When using the enzyme-labeled 1st antibody and not using 2nd antibody, try using Reagent 2 for dilution. In some cases, however, Reagent 1 works better.

6. For visualization, many users use HRP-, or AP-labeled antibody. In both cases, please watch the strength of staining or luminescence and stop the reaction. Longer reaction gives you high background or appearance of extra ba 

Protocol of ELISA

ELISA is a method to determine the amount of antigen or antibody in samples by using labeled antigen or antibody. The sandwich ELISA is most widely used, where antigen sample is applied on solid phase antibody and bound antigen is reacted by 1st antibody and visualized by labeled 2nd antibody. In some system, methods to use enzyme-labeled 1st antibody alone is also used. The way to use IMMUNO SHOT in these sandwich ELISAs are described below.

1. Antibody attachment (solid phase), blocking and the washing procedure should be done by usual method.

2. Dilute the antigen and 1st antibody with Reagent 1 of IMMUNO SHOT. The dilution factor is influenced by many factors, such as antibody species, amount of antigen, and other factors, and is important to get good sensitivity. Please refer to the supplier’s instruction to determine the best antibody concentration. Pour appropriate amount of these dilutes into each well, mix and incubate for appropriate time. Alternatively, in some method, antibody diluent is added after the addition of antigen samples.

3. Dilute the 2nd antibody with Reagent 2 of IMMUNO SHOT. The best dilution factor is influenced by many factors, such as antibody species, amount of antigen, etc.  Refer to the supplier’s instruction to determine the best antibody concentration.

4. When using the enzyme-labeled 1st antibody and not using 2nd antibody, try using Reagent 2 for dilution. In some cases, however, Reagent 1 works better.

5. For detection, many users use HRP-, or AP-labeled antibody. In both cases, please perform pre-test to determine the best reaction time. Longer reaction gives you higher background.

Trouble shooting

Trouble Cause and resolutions
Western blotting
Weak signal 1. Low antigen conc.: Use higher antigen conc.
2. Low antibody conc.: Survey best antibody conc.
3. Not enough transfer: Use higher current or longer transfer time.
4. Blocking too strong: Do not use long time blocking.
5. Too much transfer: When using nitrocellose, proteins pass though the membrane by strong transfer manipulation. Check the procedure or exchange membrane to PVDF.
Partial whitening (lumines.) 6. Too much antigen or antibody: Over signaling often suppress the luminescence and cause partial whitening of a band. Control the amount of antigen or antibody concentration.
Too many extra-bands 7. Too much higher antibody conc.: Higher antibody conc. often causes non-specific signaling. Control the antibody conc.
8. Too much antigen: Higher antigen often causes non-specific signaling. Control the amount of antigen.
9. Not enough blocking:  Some antigen and antibody have preference of blocking agents, change the blocking agents or check the blocking conditions. 
10. Not enough washing: Increase the number and time of washing.
High background 11. High antibody conc. Or too long incubation:  Reduce the antibody conc. or shorten the incubation time.
ELISA
Weak signal 1. Too low antigen or antibody conc.: Increase the concentrations.
Too strong signal 2. Too high antigen or antibody conc.: Check the antigen and antibody concentration by performing the titration.
3. Too long incubation: shorten the incubation time.
High background 4. Too high antigen or antibody conc.: check antigen and antibody cocn.
5. Not enough blocking: Some antigen and antibody preference of blocking agents, change the blocking agents or check the blocking conditions.
6. Not enough washing or too much washing: check the number and time of washing.

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.