Western Blot in Maize

Materials and Reagents

1. Protease-free pipette tips (1,000, 200 and 10 μl)
2. 1.5 ml Eppendorf tubes
3. Immuno-Blot PVDF Membrane, Roll, 26 cm x 3.3 m (Bio-Rad, catalog number: 1620177)
4. Filter paper squares, 7.5 cm x 8.4 cm
5. 12 x 12 cm Square Petri dishes
6. Transparent plastic wrap
7. X-ray films
8. Paper disposable wipes
9. Tris base (Sigma, catalog number: T1503)
10. Sodium chloride (Sigma, catalog number: S7653)
11. IGEPAL-CA-630 (Sigma, catalog number: I8896)
12. Complete^TM Protease Inhibitor Cocktail tablets (Sigma, catalog number: 11697498001)
13. PhosSTOP^TM Phosphatase inhibitor tablets (Sigma, catalog number: 4906845001)
14. DL Dithiothreitol (Sigma, catalog number: 43819)
15. Sucrose for molecular biology (Sigma, catalog number: S0389)
16. Glycerol for molecular biology (Sigma, catalog number: G5516)
17. Methanol for HPLC (Sigma, catalog number: 34860)
18. Ethanol anhydrous (Sigma, catalog number: 459836)
19. Tween 20 for molecular biology (Sigma, catalog number: P9416)
20. 30% Acrylamide/Bis Solution, 29:1 (Bio-Rad, catalog number: 1610156)
21. EDTA, Disodium Salt, Dihydrate (Sigma, catalog number: 324503)
22. Magnesium chloride (MgCl₂) (Sigma, catalog number: M8266)
23. β-mercaptoethanol (Sigma, catalog number: M6250)
24. Sodium dodecyl sulfate, SDS (Sigma, catalog number: 436143)
25. Bromophenol blue (Bio-Rad, catalog number: 1610404)
26. Ammonium persulfate for electrophoresis, ≥ 98% (Sigma, catalog number: A3678)
27. Prestained protein ladder, PageRuler 10 to 180 kDa (Thermo Fisher Scientific, catalog number: 26616)
28. Nonfat-Dried Milk (Sigma, catalog number: M7409-1BTL)
29. Bovine serum albumin (Sigma, catalog number: A3311)
30. Primary antibody Anti-NOD (Rosa et al., 2017)
31. Secondary antibody Polyclonal Anti-Guinea pig HRP-conjugated produced in Goat (Thermo Fisher Scientific, catalog number: A18769)
32. Restore Western blot stripping buffer (Thermo Fisher Scientific, catalog number: 21059)
33. Clarity Western ECL Substrate (Bio-Rad, catalog number: 1705061) (see Note 2)
34. Clarity Max Western ECL Substrate (Bio-Rad, catalog number: 1705062S) (see Note 2)
35. NBT/BCIP substrate ready to use tablets (Sigma, catalog number: 11697471001)
36. Glycine for electrophoresis (Sigma, catalog number: G8898)
37. Bovine serum albumin (BSA) (Sigma, catalog number: A9647)
38. Tris base (Sigma, catalog number: T1503)
39. TEMED (N,N,N′,N′-Tetramethylethylenediamine) for electrophoresis (Sigma, catalog number: T9281)
40. MQ water and distilled water
41. Total protein extraction buffer (see Recipes)
42. 5x Loading buffer (see Recipes)
43. 1x Tris-Glycine running buffer (see Recipes)
44. Transfer buffer (see Recipes)
45. 1x TBST buffer (see Recipes)
46. Ammonium persulfate (APS) (see Recipes)
47. Blocking solution (see Recipes)
48. Blocking solution for detection of phosphorylated proteins (see Recipes)
49. Ponceau S Staining Solution (see Recipes)
    

Equipment

1. Pipettes
2. Benchtop centrifuge

3. Vortex homogenizer
4. Thermoblock
5. Equipment for SDS-PAGE and Western blot
   Note: For SDS-PAGE, we have had success with the Bio-Rad electrophoresis equipment. For transfer, a semi dry Blotter is very good when working with proteins from 10 to 120 kDa because only 10 to 20 min at 15 V at room temperature is enough for complete transfer. When working with proteins with higher molecular weight, it is better to use wet transfer, it will take 1 h at 94 V on ice bucket for complete transfer.
6. Forceps
7. Platform rocker
8. Trans-blot Turbo Transfer (Bio-Rad) or Fast Semi Dry Blotter (Thermo Scientific)
9. X-ray film cassette and X-ray film developer machine or a Chemi-doc imager machine
10. Office scanner
    

Software

1. ImageJ (https://imagej.nih.gov/ij/)

Procedure