Mar 09, 2023
Leaf Protein Extraction for Immunoblot (Soybean, Cowpea, Tobacco)
This protocol is a draft, published without a DOI.
- 1University of Illinois at Urbana-Champaign
Protocol Citation: Steven J Burgess 2023. Leaf Protein Extraction for Immunoblot (Soybean, Cowpea, Tobacco). protocols.io https://protocols.io/view/leaf-protein-extraction-for-immunoblot-soybean-cow-cquivwue
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 09, 2023
Last Modified: August 14, 2023
Protocol Integer ID: 78442
Keywords: Glycine max, soybean, protein extraction
Abstract
This protein extraction protocol was developed for analysis of protein abundance in leaf tissue by immunoblot. It was optimized for dicot species including Glycine max.
Quantities - 450 µL protein extraction buffer (PEB) per three 13.4 mm diameter leaf disks (size #7 Humboldt Cork Borer)
Note from Agrisera: Keeping sample volumes in a range of 0.2-0.5 mL has been found to contribute to better extraction results, an upscale in volume is not recommended, if no cork borer available the suggestion is 500 µL PEB per 100 mg of plant tissue.
- Final volume ~400 µL
- Expected yield: 1.5 - 6 µg / µL
- Total yield: 6 - 24 mg protein
- Amount of total soluble protein to load per lane 30 µg
Note: when using the TissueLyser II it is recommended to use 2 mL centrifuge tubes in conjunction with 4mm beads. 1.5mL tubes are narrow at the end and samples will not grind properly if you use the 4mm beads.
Image Attribution
Image reproduced from the QIAGEN website https://www.qiagen.com/us/products/human-id-and-forensics/automation/tissuelyser-ii/#orderinginformation
Materials
- 2 mL centrifuge tubes
- Humboldt brass cork borer set (07-865-10B; Fisher Scientific)
- 13.4 mm diameter, flash-frozen leaf disks
- 4x Protein Extraction Buffer (PEB) (8 % SDS (w/v); 40 % glycerol (v/v); 0.25 M Tris HCl (pH 6.8))
Note
(from Abcam) SDS grade is important for high-quality protein separation: a protein stained background along individual gel tracts with indistinct or slightly distinct protein bands are indicative of old or poor quality SDS. This buffer is essentially the same as Laemmli buffer so proteins can be directly loaded onto PAGE gels.
Safety warnings
Perform steps with protein extraction buffer in a fumehood
Before start
Collect tissue immediately into liquid nitrogen and store at -80C. Grind per protocol "Grinding Tissue with the Qiagen Tissuelyzer".
Prepare a working solution of 1x Protein Extraction Buffer
- 1x PEB (10 mL) (2.5 mL 4x PEB; 250 µL 2-mercaptoethanol; 100 µL protease inhibitor cocktail; 7.3 mL dH2O)
Buffer Preparation
Buffer Preparation
20m
20m
Incubate 4x PEB (if stored at 4 oC) at 50 °C for 00:20:00 to re-suspended precipitated SDS in buffer.
20m
Make 500 µL 1x PEB per sample by diluting 4x stock with dH2O.
Note
It is advised to make more 1x PEB than necessary to avoid running out.
Dissolve ground powder
Dissolve ground powder
1h 3m
1h 3m
Add 450 µL 1x PEB to ground powder (3x 1cm leaf disk).
Vortex immediately (maximum speed) to mix. Approximately 00:01:00
Note
No lumps should be visible at this stage if the sample is ground well.
1m
Heat samples at 95 °C for 00:05:00 to denature proteins and inactivate proteases.
Note
Prolonged heating can cause cleavage of peptide bonds leading to artifacts. Do not delay heating after sample buffer addition as not all proteases are denatured by SDS and partially denatured peptides are sensitive to protease degradation.
5m
Vortex heated samples to shear nucleic acids (00:00:05 ; max speed)
5s
Spin samples for 00:03:00 at 10000 x g, Room temperature to pellet insoluble material
Note
the pellet should be white/light-grey an intense green color of the pellet can indicate that disruption was not optimal and extraction conditions need to be adjusted (e.g. improved grinding, or adjusting buffer volume)
3m
Transfer supernatant to a new 1.5 mL centrifuge tube and transfer to ice or store at -20 °C for up to a month
Note
Be careful not carry over debris