Anti-AtpB | Beta subunit of ATP synthase (chloroplastic + mitochondrial) (chicken antibodies)

Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts

AtpB quantitation in plant and algal samples using Agrisera Anti-AtpB global antibody and AtpB protein standard

Methodology: Plant samples are generally ground with liquid nitrogen in a mortar and pestle. The resulting powder is transferred to a plastic tube. Algal samples can be either concentrated by centrifugation or, preferably, by filtration onto glass fiber filters. Solubilization is performed in Agrisera protein extraction buffer (PEB, AS08 300) containing 0.1mg/mL PefaBloc SC (AEBSF) protease inhibitor (Roche). Disruption is most optimally obtained through flash freezing of the sample in liquid nitrogen alternated with thawing by sonication with a microtip. This process can be repeated depending on the toughness of the sample. The sample is adjusted to 50 mM dithiothreitol and heated to 70°C for 5 minutes. Samples are cooled and centrifuged briefly prior to electrophoresis.

Optimal quantitation is achieved using moderate sample loads per gel lane, generally 0.5 to 2.5 ug total protein, depending on the abundance of the target protein.

Electrophoresis and Immunoblotting: Once solubilized, the proteins can be separated electrophoretically in a number of systems. We obtain optimal results with the Invitrogen NuPAGE gel system using Bis-Tris 4-12% gradient gels. Proteins are separated in MES SDS running buffer according to the manufacturer’s recommendations at 200 V for 35 minutes. The gels are transferred to PVDF in the same apparatus, the SureLock XCell blot module, for 60 minutes at 30 V for a single gel or 80 minutes for a pair. Following transfer the blots are blocked with non-fat dry milk up to 10 % in TBS-T, for 1 h/RT with gentle agitation. The blot is incubated with primary antibody, usually at 1:25 000 to 1:50 000, for 1 h/RT (if extreme femtogram detection reagents are used) or in lower primary antibody dilution for less sensitivie reagents (mid picogram and lower).

For quantitation a relatively high primary antibody: target protein ratio gives more reliable results than immunoblots at low ratios of primary antibody:target protein.

The blot is washed extensively in TBS-T (twice briefly, once for 15 minutes and three times for five minutes). The blot is incubated with secondary antibody, for example goat anti-chicken IgY horse radish peroxidase conjugated, for 1h/RT. The blot is washed as above and developed with ECL detection reagents.

Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using the available software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max or equivalent instrument using Bio-Rad QuantityOne software. The contour tool is used to select the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude range in target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to load response curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturated response with high loads. For immunoquantitation it is critical that the target proteins in the samples and the standard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2 orders of magnitude, but the quantifiable range is narrower.

Recommended secondary antibody from Agrisera: Rabbit anti-chicken HRP conjugated or Goat anti-chicken HRP conjugated or Goat anti-chicken ALP conjugated

Recommended chemiluminescent detection reagent: AgriseraECLBright

Western Blot using IgY

Important note: Please, be aware, that often protocols used for IgG antibodies (rabbit, goat, mice) have to be optimised to get best results with IgY antibodies.

1. Block unspecific binding by washing membrane in 25 ml blocking buffer/13x13 cm filer for 2 hours at room temperature on a shaker.

* Example of a Blocking buffer (Blotto)
1x TBS (20 mM Tris/HCl, pH 7.5,150 mM NaCl, 3-5 % low-fat dried milk powder, 0.05 % Tween-20 (or Nonidet P-40)
Blotto - Limitations:
o Usually very effective however: there are complex carbohydrates in milk and these will absorb out antibodies that recognize carbohydrate determinants.
o Some IgY antibodies might recognize milk proteins (high background signal).
o 10 % nonfat dry milk might block so efficiently, and in some cases so well, that no bands of interest will be seen.
 
Blocking insufficient - alternatives to try:

Increase non-fat milk to 10-15 % 1h/RT incubation.
 

2. Wash each filter briefly, twice, then 1 x 15 minutes and 4 x 5 minutes at RT in Washing buffer. In case of still high background siganl - increase the length of a washing step even more than what is recommended above.

o Washing buffer:
1 x TBS, 0.05 % Tween-20

3. Add primary antibody to 10-20 ml of Antibody buffer (dilution range from 1:100 to 1: 30 000) and incubate the filter for 1-3 hours at room temperature (or 37°C, optional).
o Antibody buffer: 1 x TBS, 2% milk powder, 0.05 % Tween 20

4. Rinse the filter briefly twice, following by 1 x 15 and 4 x 5 minutes at RT in Washing buffer.

5. Add secondary antibody in Antibody Buffer. Use for instance rabbit or goat anti-IgY HRP conjugated. Start with the dilution at least 1 :10 000 (even higher dilutions are recommended).
Cross-reactivity signal coming from a secondary antibody can be easily checked by omitting a primary antibody in the whole procedure. Recommended secondary antibody from Agrisera: Rabbit anti-chicken HRP conjugated or Goat anti-chicken HRP conjugated or Goat anti-chicken ALP conjugated

6. Wash the filter 4 x 5 minutes in Washing buffer, followed by 1 x 15 minutes in dH2O at room temperature.

Important!
Amount of membrane washes. In case of high unspecific background, increase the time of the wash with frequent exchange of Washing buffer.
In case of high background levels, firstly test if the secondary antibody is not contributing to the background. Use a small piece of empty transfer membrane, follow the procedure above without the step with primary antibodies. Recommended chemiluminescence detection reagent in mid picogram range: AgriseraECLBright, since primary antibodies can be used in a very high dilution, often background signal will be diluted out at that step.

Using extra sensitivite development systems (above extreme femtogram) can contribute to increased background signals.
Check also: Western Blot troubleshooting

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ImmunoGold
Gellért et al. (2018). A single point mutation on the cucumber mosaic virus surface induces an unexpected and strong interaction with the F1 complex of the ATP synthase in Nicotiana clevelandii plants. Virus Res. 2018 Jun 2;251:47-55. doi: 10.1016/j.virusres.2018.05.005.

Andersson et. al (2009). Co-localization of P-glycerate kinase, P-ribulokinase, ADP-glucose pyrophosphorylase and Rubisco activase with CF1 in pea leaf chloroplasts. Plant Science 177:136-14. (immunologold)