Geoffrey Solares

How to prepare samples for Western Blot using Bio-Rad 4X Laemmli Buffer?

I need assistance verifying my calculations prior to proceeding. I have muscle homogenate (a unmarried sample to use every bit an example) at a concentration of half dozen.82mg/mL which calculates to 6.82ug/uL.

Now, the antibodies I am using recommend a range of twenty-30 ug of protein as sufficient for an adequate signal. Is 20ug, besides depression? Should I split the difference and aim for 25 ug?

Then, if I proceed with 20ug, for the purpose of verifying my calculations, I would need 2.93uL of sample per lane (all gels are run in indistinguishable). I tend to make double what I need in the outcome I have to re-run my sample and to account of loading mistake (I am multiplying this value by 4.6). The result is I need 13.48 uL sample. Using the 1:3 ratio of 4X Laemmli to sample, I require 4.5uL of 4X Laemmli.

Now, per the instructions included with the 4X Laemmli solution from Bio-Rad, "to obtain a terminal 1x concentration of 2.5% 2-mercaptoethanol (BME, 355mM): add 100uL of BME to 900 uL 4X Laemmli" <-- does this make 1x Laemmli solution?

If so, my math states I would need to add 22 uL of this solution to my sample for a loading volume of 20uL per lane in my gel. Are my calculation correct?

Thanks for any and all help!

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University of Texas at Austin

Thank you everyone. I appreciate the time to read and answer my question(southward). Accept a great rest of your weekend.

All Answers (4)

Hello in that location,

Really reductant is non present in your stock of loading buffer because it is very unstable in time. Therefore the addition of 100µL of BME to 900µL of loading buffer is just fabricated to form the existent 4x Laemmli buffer.

Hello,

There are a number of ways to make your sample. Your calculations are correct for diluting the 4x buffer to 1x with 2.v% last BME. Merely this is what I would exercise:

Dilute the 4x to 2x with water and 5% BME to make your final working sample buffer

(100 ul = 50 ul Laemmli + 5 ul BME + 45 ul water).

Yous can make any volume of buffer y'all need to yield the same proportions, depending on how many lanes you have.

Then add 10ul of this 2x buffer to 3 ul of sample + vii ul water, heat at 85C for 10 min, pulse spin when cool to pull downwards any condensate, and load all of the sample on your gel. If you have other samples with differing protein concentrations, you just make them all 10 ul with the add-on of water.

All the samples will have the same terminal volume, and y'all can just add ten ul of 2X buffer to all of them.

Always add together sample buffer with no protein to the blank wells.The sample for the blank wells would just contain 10 ul water + 10 ul 2X Laemmli, no protein.

If you want to brand extra, simply multiple all the volumes by the same amount (in your case 4.vi), load the number of lanes you need, and freeze the remainder.

Hi Geoffery, here in order to load twenty ug in twenty uL past using iv x Laemmli merely you lot need to add 3 uL of your sample (2.93 ul)+ 12 ul water + 5 ul Laemmli (BME added). Equally iv x means you need three parts of sample and i part of Laemmli. It is not the way that calculation BME in any amount will make information technology 1x. Then the x value of Laemmli is only calculated with the sample book.

Regards...

Similar questions and discussions

What 2x Laemmli Sample Buffer recipe is better?

  • Xianfeng Wang Xianfeng Wang

Up till now, there are two kinds of 2x Laemmli sample buffers:

Buffer 1) 65.8 mM Tris-HCl, pH 6.viii, 2.i% SDS, 26.three% (west/v) glycerol, 0.01% bromophenol bluish

Please refer to the original newspaper: Cleavage of structural proteins during the assembly of the head of bacteriophage T4

And BioRad, Cat# 161-0737.

Buffer two) iv% SDS, twenty% glycerol, ten% two-mercaptoethanol, 0.004% bromphenol blue and 0.125 M Tris HCl, pH approx. 6.8

Please refer to Sigma, Cat# S3401. And Cold Spring Harbor Protocols.

Buffer ane) is for optimal band resolution based on BioRad transmission.

Does anybody know the advantage of buffer 2)?

Thanks.

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