Plant Cell lysis buffer for Western and IP

Store at -20°C for up to 1 year.

PMSF (ST506) is required, but not supplied in this product. Inhibitor cocktails can also be used for better lysis results.
All procedures for sample lysis should be performed on ice or at 4°C.

Plant Sample Preparation

1. Lysis of cultured cells
a. Thaw Plant Cell Lysis Buffer for Western and IP completely and mix well. Just before use, take an appropriate amount of Lysis Buffer and add PMSF to a final concentration of 1mM. Other protease and phosphatase inhibitors can also be used if required.
b. Collect cells by centrifugation, add 100-200μl of Lysis Buffer per 0.5-1.0×106 cells. Resuspend cells by pipetting and lyse cells on ice for 2-10 min.
c. After full lysis of cells, centrifuge at 10,000-14,000×g for 3-5 minutes and take the supernatant for PAGE, Western or IP analysis.
2. Lysis of protoplasts
a. Cut tissues into small pieces and prepare the protoplasts.
b. (Optional) Transform the plasmid into protoplasts and continue incubation for 16-48 hours. Treat protoplasts as desired.
c. Centrifuge at 100-500×g to collect protoplasts.
d. Thaw Plant Cell Lysis Buffer for Western and IP completely and mix well. Just before use, take an appropriate amount of Lysis Buffer and add PMSF to a final concentration of 1mM. Other protease and phosphatase inhibitors can also be used if required.
e. Add 100-200μl of Lysis Buffer per 0.5-1.0×106 protoplasts, flick the bottom of the tube to lyse protoplasts thoroughly. There should be no obvious precipitates after lysis. For large amounts of protoplasts, dispense them into 0.5-1.0×106 protoplasts per tube for lysis.
3. Lysis of tissues
a. Cut tissues into small pieces.
b. Thaw Plant Cell Lysis Buffer for Western and IP completely and mix well. Just before use, take an appropriate amount of Lysis Buffer and add PMSF to a final concentration of 1mM. Other protease and phosphatase inhibitors can also be used if required.
c. Add 100-200μl of Lysis Buffer per 20mg of tissues. The amount of Lysis Buffer can be adjusted based on the lysis results.
d. Homogenize tissues thoroughly with a glass homogenizer or the TissueMaster Handheld Homogenizer (E6600).
e. Centrifuge at 10,000-14,000×g for 3-5 minutes and take the supernatant for PAGE, Western or IP analysis.
f. For tiny tissues, add Lysis Buffer after appropriate cutting and lyse tissues with vigorous vortex. Centrifuge and take the supernatant for subsequent analysis. This lysis method is convenient, with no homogenization needed, but the lysis result is not as thorough as that from the homogenization method.

For general Western, IP or co-IP assays, we recommend using Lysis Buffer for Western & IP (P0013) which has been extensively used with excellent performance. Lysing cells with this lysis buffer does not need ultrasonic processing. The obtained sample lysate has no gelatinous DNA clump and is ideal for subsequent studies as well as detection of phosphorylated proteins by Western.

For IP of some special proteins, RIPA Lysis Buffer or NP-40 Lysis Buffer can be attempted when Lysis Buffer for Western & IP (P0013) does not work well. If the background of IP result is high, a stronger lysis buffer such as RIPA Lysis Buffer (strong or medium) should be used. If the target protein can not be immunoprecipitated, it indicates that the lysis buffer used is too strong, and a milder lysis buffer such as RIPA Lysis Buffer (Weak) and NP-40 Lysis Buffer should be tried.

For Western blot of some proteins hard to be soluble, RIPA Lysis Buffer (Strong or Medium) or SDS Lysis Buffer can be attempted when Lysis Buffer for Western & IP (P0013) does not work well. RIPA Lysis Buffer (Strong) is also extensively used by investigators and has been cited in large amounts of articles.

For special applications where no inhibitors or a particular inhibitor is needed, P0013J or P0013K can be selected. P0013J is compatible with enzymatic activity assay and small molecule detection in most cases, but for special enzymes or small molecules, users should check the compatibility on their own. P0013J has weaker lysis capability than P0013K, but P0013J generally has better compatibility with enzyme and small molecule studies.