What is the best mycobacterial cell lysis method for identifying protein interactors using affinity purification - mass spectrometry?

18 June 2020 1 8K Report

I have cloned my gene of interest with N-terminal 1X FLAG tag in a constitutive ectopic vector and transformed in Mycobacterium smegmatis. Now i would like to pull down the interactors of the protein using Anti-FLAG M2 magnetic beads. So, what cell lysis method would be best to extract total protein?

Soundarya Josyula

sonication at 50% amplitude with EDTA free lysis buffer is suggested (Tris, SDS, tween20, DTT, PIC and Lysozyme/Dnase/Rnase). 10 cycles of sonication (one min on and one min off (on ice)) is better! U can also try it in tissue lyser as well

Badges
Science topic

Similar topics
Chemistry
Pharmacology

More Saran N.'s questions See All

Subculturing of colletotrichum capsici is not able to do please help me?

Hello all, i am working on Chilly anthracnose disease and the pathogen involved is colletotrichum capsici but the problem is occuring while subculturing, i can isolate pathogen from chilly fruit...

17 July 2024 1,610 2 View

What is the minimun number of studies for each subgroup analysis in meta-analysis?

Hello everyone, I am conducting a meta-analysis to find if x has superior effect than y on the outcome. I used the SMD method and I got a significant moderate heterogeneity of a value I2:...

09 June 2024 8,446 3 View

Effect size to check if x is superior than y... How to calculate it? within or between?

Hello everyone, I am conducting a systematic review to see if treatment x is superior in its effect than treatment y. Since most the studies I found did not report interaction effect, I was...

24 May 2024 6,130 2 View

What are recent UWB Antennas ?

I require recent UWB antennas manufactured

15 May 2024 3,888 1 View

Which is the best free software for RAPD data analysis?

I am searching for a free best software for RAPD data analysis and phylogenetic tree construction

09 May 2024 7,017 3 View

Is there a way to combine CFA with linear mixed model taking into account random effects?

I try to explaim better. I have the scores for 10 items. Each item score are decided between the accordance of two persons (say, e.g., a therapist and a patient). Therefore, there is much more...

24 April 2024 4,563 6 View

How do I interpret Hedgs g sign? How do I calculate effect size when only median with CI is reported ?

Hello everyone, I am writing a systematic review about if X experimental group has superior effect than Y comparison group on outcome. Please note that the lower the scores of the outcome, the...

19 April 2024 1,812 1 View

Methods to calculate Shannon Index?

I have three field margin types (grassy/shrubby/tree) and each type have 5 sites. I need to calculate Shannon index for insect counts from each field margin type.... Q1. In my case how should I...

19 April 2024 681 6 View

How to fix the error during importing Gas Phase reaction, surface reactiona and thermodynamic data Chemkin file in fluent?

Hello All, Greetings The issue I am encountering pertains to the Chemkin files to be uploaded into the Ansys Fluent software. Unfortunately, I am encountering persistent errors, and it seems the...

04 April 2024 8,189 0 View

How to find Functional role of insects?

Respected all Is there is any database which classify insects according to their guild (feeding habit) and Ecological/functional roles like predator, parasitoid, scavenger, pollinator. Please let...

03 April 2024 3,485 5 View

Is there a problem with my RNA pellet?

Hello, I am currently having problems with RNA extraction. I am using mouse liver (C57BL6J), and I have extracted RNA from mouse liver before. Before this experiment, my final RNA pellets were...

11 August 2024 7,082 3 View

Can you connect an HPLC to a Mass Spec only at a certain time point?

Can anyone explain this method? Especially the last statement where it says only at 1.5 to 2.5mins was the MS/MS connected to the UPLC. How is that possible, is it a feature in this specific...

11 August 2024 8,141 3 View

Why Do TDS and EC Increase with Larger Wastewater Volumes, While BOD and COD Decrease?

I have carried out MFC experiments on three different volumes, 50, 500 and 1000 mL of wastewater. Results after MFC treatment shows that TDS and EC are more in larger volumes of water i.e. TDS and...

09 August 2024 9,621 0 View

Could it be a cell culture contamination?

Hello everyone! I observed in my culture (htert-RPE1 cells) an orange- red particle at the bottom of the dish. It is visible to the naked eye as a very very small red dot. Could it be a...

09 August 2024 2,824 3 View

GC-MS retention index prediticon?

Hello experts, Does anyone know any free software about retention index prediction ?

08 August 2024 7,403 2 View

RNA Extraction Using Hot Borate Method No Longer Working?

I've been performing RNA extraction on cotton petiole tissue for a few months now using the method described in the following paper, a derivative of the typical hot borate method...

08 August 2024 9,882 2 View

How to calculate CCS for Sodiated adduct ions and Multiply Charged Ions?

I'm currently working on calculating the collision cross section (CCS) for various ions, and I'm facing challenges when dealing with sodiated and multiply charged ions. Most of the resources I’ve...

08 August 2024 8,329 0 View

Is there any way to quantify bacterial and fungal cells in their mixed culture?

I am working in fungal fermentation of soybean meal and there is bacterial growth in them at times. I am trying to quantify fungal cell counts and bacterial cells; but I haven't been able to do at...

07 August 2024 7,535 4 View

How to normalize and take the significance of the MTT OD values with 3 replicates for the same cell-line?

Hi, I have a question about normalizing the MTT OD values for doing the statistical analysis. So, if we have 3 different plates and we call them 3 different replicates, so, first we would...

07 August 2024 8,106 4 View

Why activated CAR-Jurkat cell could not kill targets?

Previously when I co-coluture anti-CD19(FMC63) CAR-Jurkat with Raji with E:T=5:1, Jurkat can eliminate Raji in 24h. However, when I test another CAR construct, although I can dectect totally CD69...

06 August 2024 641 2 View