Home >> News

Various Sample Processing and Preservation of Western Blot

May 18, 2022

Before dealing with Western Blot (WB), sample handling and preservation are crucial. For your information, this chapter organizes the methods of handling and preserving various samples of WB, such as cell, tissue, plant and bacterial samples.

Cell samples

1. Suspended cells

1) Collect the cells and culture fluid together into a 15ml or 50ml centrifuge tube, centrifuge at 1500rpm for 3min and discard the supernatant.

2) Add PBS and blow gently, centrifuge at 1500rpm for 3min and discard the supernatant.

3) Repeat 3 times, discard the supernatant and freeze at -80 ℃ or in liquid nitrogen for use.

2. Adherent cells

1) Trypsin digestion: After digestion with trypsin, the cells were collected into 15 ml or 50 ml centrifuge tubes, centrifuged at 1500 rpm for 3 min, and the supernatant was discarded.

2) Add PBS and blow gently, centrifuge at 1500rpm for 3min and discard the supernatant.

3) Repeat 3 times, discard the supernatant and freeze at -80 ℃ or in liquid nitrogen for use.

3. Using cell scrapers to collect cells

For certain proteins (i.e., sensitive to trypsin or experimentally specific requirements, such as E-Cadherin which can’t be easily digested with trypsin for WB detection), cells were scraped off directly with a cell scraper.

1) Collect the scraped cells into a centrifuge tube, centrifuge at 1500 rpm for 3 min, and discard the supernatant.

2) Add PBS and blow gently, centrifuge at 1500rpm for 3min and discard the supernatant.

3) Repeat 3 times, discard the supernatant and freeze at -80 ℃ or in liquid nitrogen for use.

Animal tissue samples

1) Remove suitable tissue samples and rinse with pre-chilled PBS or saline to remove as much residual blood as possible, and aspirate the excess PBS with filter paper.

2) Divide the tissues into approximately 100 mg size (adjusted according to the experimental purpose), wrap them separately in tinfoil or pack them in lyophilized tubes and store them at -80 ℃ or in liquid nitrogen.

Plant samples

After obtaining the plant samples to be tested, the soil was rinsed with distilled water, the excess water is sucked out with filter paper and stored at -80°C or in liquid nitrogen in appropriate sizes.

Bacterial samples

1) After the bacterial culture is completed, collect the bacteria by centrifugation at 10000rpm for 5min, add PBS and blow, centrifuge at 10000rpm for 5min and discard the supernatant.

2) Repeat 3 times, discard the supernatant and freeze at -80 ℃ or in liquid nitrogen for use.

Tips: When you are detecting phosphorylated protein from the samples above, it is recommended that after you extract protein from fresh samples, you’d better added Loding buffer to the extracted protein and boiled for 10min. It should be stored at -20 ℃ in the short term, and -80 ℃ in a long term. Please finish it within 3 months, because the samples are easy to dephosphorylate phosphorylated protein and can’t detect phosphorylated bands.

Caution:

(1) Samples for extraction of cytoplasmic and cytosolic proteins should be as fresh as possible; frozen samples may yield less cytosolic protein.

(2) The samples should be stored at -80℃ or liquid nitrogen, so as to avoid repeated freezing and thawing. It is recommended that they should be tested as soon as possible.

(3) Dry ice or liquid nitrogen should be used to transport samples over long distances.


News & Event
Various Sample Processing and Preservation of Western Blot
Various Sample Pr... May 18, 2022

Paper Reward Plan
Paper Reward Plan Oct 17, 2016

Copyright © Shanghai YL Biotech Co., Ltd All Rights Reserved

Technical Support:

Contact Us
  • Tel: +86 021 5772 3955
  • Fax: +86 021 5772 3955
  • Orders for consulting: export@ylbiont.com
  • Technical consulting: william@ylbiont.com
  • Add: 3577 Jinke Road, Zhangjiang Town, Pudong New Area, Shanghai
Top