Western blotting

We offer products for all the steps in your western blotting:

• Sample preparation
• SDS-PAGE
• Transfer
• Blocking
• Antibody incubations
• Washing
• Detection 

Western blotting

Sample preparation

To prepare samples for western blotting (WB) analysis, it is recommended that chemical and physical methods are used to disrupt membranes and ensure cell lysis and the release of proteins of interest. For sample lysates from tissues, a homogenization step is recommended in most cases, and a protease inhibitor cocktail is usually added the the lysis buffer prior to homogenization. Lysis buffers contain varying degrees of detergents, salts, and enzymatic inhibitors (such as phosphatase or protease inhibitors) to break apart cells, solubilize proteins, and prevent degradation. 

We offer several sample preparation buffers and sonication devices. 

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SurePAGE precast gels

SurePAGE Bis-Tris gels from GenScript are high-performance precast mini polyacrylamide gels that separate a wide range of protein sizes by electrophoresis. The gels are cast in a neutral pH buffer that minimizes polyacrylamide hydrolysis and increases gel stability. They run at neutral pH which minimizes protein modification compared to Tris-glycine gels.

Proteins are separated on SurePAGE either using MOPS or MES running buffer. We recommend using MOPS buffer for protein sizes that are >30kD and MES for proteins that are <30 kD. And we recommend using 4×LDS sample buffer (#M00676) as the sample loading buffer.

SurePAGE precast gels
Cat.no. % Acrylamide Wells no. Well volume Quantity
M00652, M00653, M00654 4-12% 10, 12, 15 80, 60, 40 μl 10 gels
M00655, M00656, M00657 4-20% 10, 12, 15 80, 60, 40 μl 10 gels
M00658, M00659, M00660 8-16% 10, 12, 15 80, 60, 40 μl 10 gels
M00661, M00662, M00663 8% 10, 12, 15 80, 60, 40 μl 10 gels
M00664, M00665, M00666 10% 10, 12, 15 80, 60, 40 μl 10 gels
M00667, M00668, M00669 12% 10, 12, 15 80, 60, 40 μl 10 gels

Compatible Gel Tanks:
Bio-Rad Mini-PROTEAN® II & 3*
Bio-Rad Mini-PROTEAN®Tetra System*
Invitrogen Novex XCell I, II, & Surelock® (Use GenScript Gel Tank Adapter Plates)
LONZA PAGEr® Minigel Chamber
Hoefer Mighty Small (SE 260/SE 250)
Hoefer Tall Mighty Small (SE 280)

*please reverse the gasket, see manual for detail
Prices and ordering

Prices are available in our webshop. You may also contact us by e-mail for price information and ordering.

Blocking

Nonfat dry milk

Premium quality nonfat dry milk for use as a blocking agent during Western blotting to block non-specific antibody binding and reduce background signal.

9999S Nonfat dry milk, 250 grams Datasheet
Animal-Free Blocking Solution (5X)

Animal-Free Blocking Solution (5X) is designed for use as a blocking reagent in chromogenic immunohistochemical assays (IHC) and western blotting. This plant-based product contains no animal-derived proteins and can be used to decrease background in place of normal goat serum, BSA, or non-fat dry milk.

15019S Animal-Free Blocking Solution (5X), 25 ml Datasheet
15019L Animal-Free Blocking Solution (5X), 250 ml Datasheet
BSA

Ultra pure bovine serum albumin (BSA) is used as a reagent during Western blotting. BSA can also be used in other protocols that involve antibody binding including ELISA, DELFIA, flow cytometry and immunofluorescence/immunocytochemistry.

9998S BSA, 50 grams Datasheet
Prices and ordering

Prices are available in our webshop. You may also contact us by e-mail for price information and ordering.

Loading control antibodies

You may use loading control antibodies to determine equal loading of the wells, as well as the quality of your samples. This is critically important to give you the best conditions when you shall interpret your western blot results. Proteins used as loading controls are typically selected for their ubiquitous expression across all cell lines and tissues as well as their abundance. Selecting the right loading control antibody is determined by the molecular weight of the other targets being studied, subcellular fraction of the sample, and the cell and tissue types being evaluated.

You need to trust that your loading control is sensitive and specific to its target protein. If your loading control is questionable, it can create doubt about all of your results. Use loading controls you trust for the most reliable experimental results. 

We recommend the loading control antibodies from Cell Signaling Technology (CST):

β-Actin 45 kDa Whole Cell Lysate GAPDH 37 kDa β-Tubulin 55 kDa α-Tubulin 52 kDa
Vinculin 124 kDa α-Actinin 100 kDa Cyclophilin B 19 kDa Histone H3 17 kDa Nuclear fraction
HDAC1 62 kDa COX IV 17 kDa Mitokondriell fraksjon VDAC 32 kDa SDHA 70 kDa

Loading control antibody sampler kits


CST offer a selection of sampler kits with small amounts of selected loading control antibodies. These Loading Control Antibody Sampler Kits provides an economical means to detect a variety of housekeeping proteins. They contain enough primary and secondary antibodies to perform two western blot experiments. 

5142S Loading Control Antibody Sampler Kit Datasheet
9774S Loading Control Antibody Sampler Kit (Mouse) Datasheet
4670S Loading Control Antibody Sampler Kit (HRP Conjugate) Datasheet
Prices and ordering

Prices are available in our webshop. You may also contact us by e-mail for price information and ordering.

Protein ladders

The selection of protein ladders on the market is huge. There are ladders for all types of protein separations.

Here we show some of the ladders we offer from our suppliers. 

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Protein ladders

Detection

To observe a maximum signal on your western blot (WB) you need detection reagents with the quality and strength to enhance a weak signal and reduce background staining. 

Cell Signaling Technology (CST) offer three types of chemiluminescent detection reagents for WB: 
 

SignalFire™ ECL Reagent

SignalFire™ ECL Reagent is an enhanced chemiluminescent substrate capable of detecting picogram amounts of protein by western blot analysis. Compared to other luminol-based systems, this reagent provides a more robust signal and extended duration of signal output. 

6883 SignalFire™ ECL Reagent Datasheet
SignalFire™ Plus ECL Reagent

SignalFire™ Plus ECL Reagent is a highly sensitive chemiluminescent substrate capable of detecting low picogram amounts of protein by western blot analysis. This reagent has an extended duration of signal output and can last several hours after blot exposure. This allows for multiple exposures with either film or a digital imaging system. The strong signal output makes it possible to detect low abundance proteins, conserve reagents, and have short exposure times.

12630 SignalFire™ Plus ECL Reagent Datasheet
SignalFire™ Elite ECL Reagent

SignalFire™ Elite ECL Reagent from Cell Signaling Technology (CST) is an ultra sensitive chemiluminescent substrate capable of detecting femtogram amounts of protein by western blot analysis. SignalFire™ Elite ECL Reagent is compatible with both film and digital imaging systems. The extremely intense signal output allows detection of very low abundance proteins, conservation of reagents, and short exposure times.

SignalFire™ Elite ECL Reagent requires approximately ten-fold less Anti-rabbit IgG, HRP-linked Antibody #7074 or Anti-mouse IgG, HRP-linked Antibody #7076 than traditional ECL reagents. Limiting the amount of HRP exposed to the membrane prevents high background, oversaturation of the target protein signal, or false negative results. Other HRP-conjugated antibodies, including HRP-conjugated primary and anti-biotin-HRP antibodies, should be diluted similarly. Dilution of secondary antibody from alternative vendors may need to be optimized. Titration of lysate and primary antibody concentration is recommended to achieve optimal signal-to-noise ratio.

12757 SignalFire™ Elite ECL Reagent Datasheet
Prices and ordering

Prices are available in our webshop. You may also contact us by e-mail for price information and ordering.

Contact person: Christine Vimo

Christine has a bachelor`s degree in biology and a master`s degree in molecular biology. Contact her if you have any questions about antibodies or the products within the areas western blotting, IHC, IF and flow, and anything you should need for your molecular biology experiments. 

christine@bionordika.no

Contact person: Christine Vimo