KeeSiang Lim

13 Questions 28 Answers 0 Followers

Questions related from KeeSiang Lim

The best culture condition for Kit-225

I am currently culturing the T-CLL, Kit-225 cell line. My culture condition is RPMI 10% FCS with 20ng/ml IL2 (R&D, about 420IU/ml). Cells look viable but proliferation rate is slow. Is there...

06 June 2017 6,510 6 View

How can I overcome such problematic melt curve in qPCR?

I am facing problem in qPCR analysis. The file attached shows the melt curve of GAPDH. In my setting, my template was 100ng and primer concentration 500nM in 20ul. Cycling number was 40 cycles. In...

01 January 2016 5,739 5 View

How to achieve better amplification in qPCR?

I am currently comparing ULBP-4 expression in cancer cells. The method I used for qPCR as follow: a) cDNA synthesis = 1ug of RNA in 20ul b) I diluted the cDNA 10X prior to qPCR c) I used 4ul of...

10 October 2015 6,337 9 View

How might I ensure RT-PCR consistency ?

I have run 3 times of RT-PCR for one of my sample. However, fold changes always inconsistant in these three attempts. What is the possible factors contribute to this inconsistancy?

07 July 2015 6,602 5 View

Why is my western blot black and without bands?

I am currently doing western blot for p-Akt. Below is my protocol: -- Blocking = RT, 2 hours with 5% BSA -- Primary antibody = p-Akt (1:2000), overnight, RT -- Secondary antibody = Anti rabbit...

05 May 2015 6,215 9 View

What is the difference between non-fat milk and BSA for primary antibody in western blot?

Some protocols mention primary antibodies should be diluted in 5%BSA whereas some protocols mention primary antibodies should be diluted in 5% non-fat milk. What is the difference? What is the...

05 May 2014 436 20 View

Is serum starve important for TGF-beta induced EMT and EGF induced EMT?

Many publications mention that cells were serum starved for 24 hours before adding TGF-beta or EGF to induce EMT. After serum starved, is it possible to treat cells with EMT-inducers in RPMI 5%...

05 May 2014 7,787 10 View

Do you know where I can find cancer genomic data on kyse50, kyse 110, TE2, and TE3?

I have found that cbioportal is a good website to study molecular expression level of certain proteins in certain cancer cell lines. Unfortunately, some cell lines are not in the database. Where...

04 April 2014 1,058 4 View

How can I improve my western blot results for the optimal dilution for a Snail-1 antibody?

I am currently comparing snail-1 expression in esophageal cancer cell lines. My protein load was 30-40 ug, the primary snail-1 antibody (cell signaling) was 1:1000, and the secondary anti-rabbit...

03 March 2014 8,714 17 View

How to reduce cell clumping after cells have been treated with enzyme-free dissociation buffer?

Does anyone know how to reduce cell clumping after cells are treated with enzyme-free dissociation buffer? Is autoMACs rinsing solution (contains PBS and EDTA) helpful to prevent cell clumping in...

03 March 2014 5,301 6 View

Does anyone know the concentration of IL-2 that is sufficient to support resting NK cells to survive without activated to IL-2 NK cells?

see above

03 March 2014 4,931 0 View

Which scale is better for the Measurement of Fluorescent Intensity?

Which scale is better, Linear scale or log scale?

03 March 2014 7,308 7 View

Can anyone help: my western blot result of a snail protein is higher than 30 KD?

The supplier COA stated that the snail protein is about 30KD. I ran the electrophoresis at 200V, 125mA for 45mins. I used 4-12% Tris Nu-PAge Gel. Why is the snail protein more than 30KD?

03 March 2014 8,135 7 View