How to determine which Sample Buffers, Running Buffers, and Transfer Buffers to use per Western Blot experiment?

18 December 2017 1 6K Report

I'd like to be methodical in deciding which buffers to use per Western Blot Experiment. A thorough system of deciding which combination of buffers to use for each WB experiment would be helpful. Thanks!

Laura Kytovuori

Hi!

I have been using NuPAGE-gels from Life Technologies and they have different buffers for different separation range (the manual contains gel migration chart in different buffers with different percentage of the gels). The manual is very comprehensive with all the recipes and the reagents have been working very well in my experiments. Just google the NuPAGE manual. Good luck!

Badges
Science method

More Randy Strauss's questions See All

I can detect my protein but the encoding DNA sequence has stop codons within. Should I ignore the stop codons?

I constructed a synthetic DNA library (scFv, VH-VL orientation) with a 3' reporter and 8x histag. I cloned and expressed this gene following which I performed an ELISA. The ELISA results suggested...

19 February 2021 7,006 5 View

How would a 'fair trade' version of a marketplace app like Uber Eats work?

(Note: ResearchGate questions does not have any comments feature. I am unable to directly respond to individual answers below because there is no commenting feature available. Please do not ask...

23 September 2020 5,727 2 View

Which scholars, pedagogues, and projects are carrying forward the legacy of arts educator Elliot Eisner (1933-2014)?

13 September 2020 3,664 4 View

Why my PCR product (band) gets smaller with every round of reamplification?

I use the same primer set and same conditions. The first PCR product from cDNA gave about 800 bp. Reamplification of the template gave 700 bp. A second amplification gave 600 bp. Interestingly, I...

24 February 2020 4,346 8 View

Help with Gibson reaction components calculation please?

An insert length of 811bp (17ng/ul) and digested vector size of 5.6kb (35ng/ul). --100ng vector amount--. Am I right in using the following respective volumes of insert and vector? 2.5ul and...

06 May 2019 7,036 3 View

Does anyone know how to calculate the sample size in an "in vitro" study using ATCC bacterias?

24 September 2014 7,051 12 View

What is a good control to measure gene expression levels in Synechocystis PCC6803?

I know in Arabidopsis people will often use something like tubulin, which is constitutively expressed, as something to normalize the amount of gene expression. I've seen people use 16s rRNA for...

13 March 2013 1,668 1 View

Graphene domain size

I am interested in calculating graphene domain size values. I have reading that the D and G Raman band intensities ratio (ID/IG) can be used to calculate it. What are the limitations of this...

09 December 2012 370 3 View

What is the TiO2 band gap type?

When we say generally TiO2 band gap energy, does it mean direct or indirect band energy? I am looking for some papers and if it's possible to find both possibilities!

26 August 2012 5,244 20 View

Buffer to disrupt platelet alpha granules for PF4 quantification?

Hello! I want to quantify by ELISA the secreted (from platelets poor plasma) and the non-secreted (from platelet lysate) PF4 before and after TRAP stimulation. I will use the ELISA from R&D...

03 March 2021 1,499 2 View

Buffer Exchange using column packed with sephadex G-25 effect on protein concentration?

Hello, If i am doing a buffer exchange for an antibody of 1mg/mL, does the elution lose protein in the process of buffer exchange? For example, if i flow through 500 uL of 1mg/mL sample, and...

03 March 2021 6,299 3 View

New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before?

Question to you and THEM, the New Journal, "Integrative Psychological and Behavioral Science" -- do you not know, and have you not seen, this done before? There appears to be a core problem for...

02 March 2021 3,024 2 View

Which is a lucid standard comprehensive book to guide on psychological scale construction and its standardization?

02 March 2021 7,082 3 View

How optimize the Size Exclusion Chromatography?

Hello! I'll do a size exclusion chromatography, but I only have an open column, and I'll perform the peptide extraction from yeast, using buffer lysis (sodium phosphate 50 mM/NaCl 30 mM/DNAse and...

01 March 2021 2,215 2 View

LOAD DISPLACEMENT CURVES Stiffness increasing in ANSYS non linear analysis. HOW CAN I CORRECT IT?

Hi. I am doing non linear FE analysis using a new material beam under flexural 2 point loading using ANSYS. I have defined a bilinear material model. The solution has converged. In results I am...

01 March 2021 9,911 3 View

Why are my bands weird in this Agarose gel?

Can someone please give me some possible things that could go wrong? Here is my recipe: 0.5g Agarose 50 mL of TAE 1x 1 uL ethyl bromide. Gel was run at 100V for 1 hour. The buffer used is also TAE.

01 March 2021 9,952 3 View

What is recommended for solid domain in ANSYS FLUENT?

Should I mesh the solid domain ? or should I use wall thickness? or Shell conduction? for Heat Transfer between two fluids of a Heat Exchanger. what is recommended?

01 March 2021 9,785 3 View

What do I do with DLS peaks that are from the buffer?

I'm looking at the aggregation of my protein sample using DLS. Unfortunately, my buffer (20mM HEPES) also results in a set of peaks. These are at approximately 1 and 1000 d.nm. The lowest peak...

01 March 2021 9,015 2 View

How to choose ANN number of hidden layers and nodes?

Hello, Could you please share any interesting research explaining how to choose the number of hidden layers and nodes per layer in case of regression problems using ANN? Thank you, any help would...

01 March 2021 6,200 3 View