Blotting Techniques

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Imaging system to use instead of the X-ray film with a Southern blot, which employed the DIG High Prime DNA Labeling and Detection Starter Kit II?

I will do Southern blot using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). Somebody can recommend me an imager or imaging system to observe the results, I don't want to...

02 February 2017 4,857 0 View

Precellys empty tube alternatives

Is there anyone using Bertin's Precellys homogenizer? I want to ask whether there is cheaper alternative for empty 7ml screw cap tubes than what they provide? It is very costly to use them...

02 February 2017 1,213 5 View

32P autoradiography detection on amersham hyperfilm ECL ?

Dear all, We retrieved a box of amersham hyperfilm ECL films and I was wondering if this kind of film is able to detect 32P autoradiography (southern and northern-blotting) ? Does anyone already...

02 February 2017 3,454 4 View

Western band unbalanced and not straight, does somebody know the reason?

I have repeated the western for several times, but it shows the same problem. It seems that some bands are squeezed(2 and 5 in the first picture, 2 and 4 in the second picture). I use the...

02 February 2017 6,800 8 View

Western band unbalanced and not straight, does somebody know the reason?

I have repeated the western for several times, but it shows the same problem. It seems that some bands are squeezed(2 and 5 in the first picture, 2 and 4 in the second picture). I use the...

01 February 2017 7,437 7 View

Higher than expected band in western blot: suggestions?

I'm currently doing a western blot to identify smooth muscle actin in cardiac myofibroblast cells. Initially when I did the westerns I use to get nice strong clear band at the expected 43kDa...

01 January 2017 2,057 5 View

Why won't my high range ladder separate?

I'm running gel electrophoresis for bacteria DNA. I used GeneRuler High Range DNA Ladder, ready-to-use. However, when I run gel electrophoresis, the DNA ladder isn't separating well. According to...

12 December 2016 9,980 5 View

When performing a semi-dry Western Blot transfer--how important are the sizes of filter papers and membrane, as long as the gel fits on them?

As long as gel has the right thickness of filter above and below it, and the nitrocellulose or PVDF membrane covers my gel, am I just as fine as if everything was cut to the exact same size? If,...

12 December 2016 9,885 1 View

Why won't my high range ladder separate?

I'm running gel electrophoresis for bacteria DNA. I used GeneRuler High Range DNA Ladder, ready-to-use. However, when I run gel electrophoresis, the DNA ladder isn't separating well. According to...

07 December 2016 380 4 View

When performing a semi-dry Western Blot transfer--how important are the sizes of filter papers and membrane, as long as the gel fits on them?

As long as gel has the right thickness of filter above and below it, and the nitrocellulose or PVDF membrane covers my gel, am I just as fine as if everything was cut to the exact same size? If,...

06 December 2016 8,137 1 View

SirT1 Western blot antibody?

We want to measure protein expression levels of SirT1 in mouse tissue (heart, skeletal muscle, liver) via Western blot. Unfortunately, no antibody has worked so far. Can someone recommend a...

11 November 2016 8,313 2 View

What should be the transfer medium for Northern?

I'm using DIG Northern starter kit by Roche. I'm trying for weeks and following the protocol (transfer in SSC 20x over night, but i tried also the trasfer in SSC 10x, the membrane is a Nylon...

11 November 2016 6,200 8 View

Could ROCHE Hybridization Buffer (cat. 11717472001) be directly used as Prehybridization and Hybridization solution for Southern Blot?

Long time ago, I bought ROCHE Hybridization Buffer (cat. 11717472001), now I want to do the SB assay, but i can't find any instructions about this buffer. So I wonder if this buffer was...

18 August 2016 1,066 3 View

Could ROCHE Hybridization Buffer (cat. 11717472001) be directly used as Prehybridization and Hybridization solution for Southern Blot?

Long time ago, I bought ROCHE Hybridization Buffer (cat. 11717472001), now I want to do the SB assay, but i can't find any instructions about this buffer. So I wonder if this buffer was...

08 August 2016 4,812 4 View

I want to resolve mRNA on a northern blot, with the fragment sizes 1230 and 1540. which % of Agarose gel should i use, runtime and Voltage?

i think it should be between 1% and 2%?

08 August 2016 9,725 3 View

I want to resolve mRNA on a northern blot, with the fragment sizes 1230 and 1540. which % of Agarose gel should i use, runtime and Voltage?

i think it should be between 1% and 2%?

04 August 2016 3,371 3 View

S it necessary to check by Southern Blot except for the PCR and sequencing test?

Dose anyone generate Knock-in and Knock-out mouse by CRISPR-Cas9 system? I constructed knock-in and knock-out mouse by CRISPR-Cas9 system?should i use southern blot technique to screen? is it...

10 June 2016 5,036 1 View

Why can't I detect p-Akt bands in my western blots?

I am looking for phospho-Akt (serine 473) in rat heart tissue samples using western blots. I have done trouble shooting for weeks but still nothing. At first we thought it was our antibodies that...

07 June 2016 5,140 5 View

S it necessary to check by Southern Blot except for the PCR and sequencing test?

Dose anyone generate Knock-in and Knock-out mouse by CRISPR-Cas9 system? I constructed knock-in and knock-out mouse by CRISPR-Cas9 system?should i use southern blot technique to screen? is it...

06 June 2016 8,036 1 View

Why can't I detect p-Akt bands in my western blots?

I am looking for phospho-Akt (serine 473) in rat heart tissue samples using western blots. I have done trouble shooting for weeks but still nothing. At first we thought it was our antibodies that...

06 June 2016 3,095 6 View

Mold Extract WesternBlot using Human Sera as 1st Antibody?

I am trying to detect a former mold exposure of patients via WesternBlot using their sera. For this purpose, I tried out the following setup:Samples: gamma-inactivated Aspergillus niger,...

06 June 2016 3,206 2 View

Does anyone have any suggestions for which GLUT antibodies to use for Western Blots against GLUT3, GLUT4 and maybe even GLUT1?

I want to quantify the amount of GLUT3, GLUT4 and maybe GLUT1 in the HPC of rats using Western Blotting. If anyone has worked specifically with these antibodies and has one that has given...

05 May 2016 7,821 3 View

For what reason , the ladders transfer to membrane and it is visible in the film at the end but my sample band isnt visible(southern blot)?

my ladders transfer and they are visible in the film but the sample bands cant be seen in the film . I use labeled probe which labeled by me and I used roche dna labelin kit . please see the...

04 April 2016 373 7 View

Is there a way to recover an antibody that is in milk that has congealed?

I am western blotting and want to use and old aliquot of an antibody we no longer have. The milk had NaN3 in it so it's not sour.

04 April 2016 4,117 3 View

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